Soluble cd33 for treating myelodysplastic syndromes (mds)

ABSTRACT

Disclosed are compositions and methods for treating disease or condition caused or exacerbated by S100A9 activity, such as myelodysplastic syndromes (MDS) using a composition comprising an effective amount of a CD33/S100A9 inhibitor.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 16/451,525, filed Jun. 25, 2019, which is a divisional applicationof U.S. application Ser. No. 14/902,719, filed Jan. 4, 2016, which is anational stage application filed under 35 U.S.C. § 371 ofPCT/US2014/045444 filed Jul. 3, 2014, which claims benefit of U.S.Provisional Application No. 61/843,274, filed Jul. 5, 2013, U.S.Provisional Application No. 61/930,798, filed Jan. 23, 2014, U.S.Provisional Application No. 61/931,366, filed Jan. 24, 2014, and U.S.Provisional Application No. 61/978,009, filed Apr. 10, 2014, which arehereby incorporated herein by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government Support under Grant No. CA131076and Grant No. AI056213 awarded by the National Institutes of Health. TheGovernment has certain rights in the invention.

SEQUENCE LISTING STATEMENT

A Sequence Listing conforming to the rules of WIPO Standard ST.26 ishereby incorporated by reference. Said Sequence Listing has been filedas an electronic document via PatentCenter encoded as XML in UTF-8 text.The Sequence Listing.xml file, identified as “10110-061US3 2023_07_17Sequence Listing.xml” is 16,127 bytes and was created on Jul. 17, 2023.

BACKGROUND

Myelodysplastic syndromes (MDS) are hematopoietic stem cell malignancieswith a rising prevalence owing to the aging of the American population.MDS comprise a group of malignant hematologic disorders associated withimpaired erythropoiesis, dysregulated myeloid differentiation andincreased risk for acute myeloid leukemia (AML) transformation. Theincidence of MDS is increasing with 15,000 to 20,000 new cases each yearin the United States and large numbers of patients requiring chronicblood transfusions. Ineffective erythropoiesis remains the principaltherapeutic challenge for patients with more indolent subtypes, drivenby a complex interplay between genetic abnormalities intrinsic to theMDS clone and senescence dependent inflammatory signals within the bonemarrow (BM) microenvironment. Although three agents are approved for thetreatment of MDS in the United States (US), lenalidomide (LEN)represents the only targeted therapeutic. Treatment with LEN yieldssustained red blood cell transfusion independence accompanied by partialor complete resolution of cytogenetic abnormalities in the majority ofpatients with a chromosome 5q deletion (del5q), whereas only a minorityof patients with non-del5q MDS achieve a meaningful response,infrequently accompanied by cytogenetic improvement. Although responsesin patients with del5q MDS are relatively durable, lasting a median of2.5 years, resistance emerges over time with resumption of transfusiondependence.

SUMMARY

It is shown herein that CD33⁺ myeloid-derived suppressor cells (MDSCs)specifically accumulate in the BM of MDS patients and impairhematopoiesis through a mechanism that involves S100A9 as an endogenousligand for CD33 initiated signaling. Therefore, disclosed arecompositions and methods for treating MDS that generally involveadministering an effective amount of a CD33/S100A9 antagonist to inhibitactivation of MDSCs. For example, the method can involve administeringto the subject a therapeutically effective amount of a compositioncomprising an agent that binds and sequesters S100A9.

In some embodiments, the CD33/S100A9 antagonist binds and sequestersendogenous S100A9 and inhibits its binding to CD33 receptor on MDSCs.Therefore, in some embodiments, the CD33/S100A9 antagonist is a moleculecontaining a S100A9-binding domain. For example, the CD33/S100A9antagonist can be a chimeric fusion protein comprising the ectodomain ofCD33, Toll like Receptor 4 (TLR4), Receptor for Advanced Glycation EndProducts (RAGE), or a combination thereof. This ectodomain of CD33,TLR4, and/or RAGE can be any fragment of CD33, TLR4, and/or RAGE capableof binding S100A9. For example, in some cases the CD33 ectodomaincontains only the variable region of CD33.

Therefore, disclosed is a recombinant fusion protein comprising animmunoglobulin Fc region; and one, two, three, or more of anextracellular domain of human CD33, extracellular domain of TLR4,extracellular domain of RAGE, or a combination thereof that binds S100A9protein and is linked by a peptide bond or a peptide linker sequence tothe carboxy-terminus of the immunoglobulin Fc region. The fusion proteincan further contain a biotin acceptor peptide that can be biotinylatedwith biotin ligase (BirA) in the presence of biotin and ATP.

In some embodiments, the fusion protein comprises a formula selectedfrom the group consisting of:

-   -   eCD33-eTLR4-Fc,    -   eCD33-eRAGE-Fc,    -   eTLR4-eRAGE-Fc,    -   eRAGE-eTLR4-Fc,    -   eCD33-eTLR4-eRAGE-Fc,    -   eCD33-eRAGE-eTLR4-Fc,    -   eTLR4-eCD33-eRAGE-Fc,    -   eRAGE-eCD33-eTLR4-Fc,    -   eTLR4-eRAGE-eCD33-Fc, and    -   eRAGE-eTLR4-eCD33-Fc,    -   wherein “eCD33” is the extracellular domain of human CD33,    -   wherein “eTLR4” is the extracellular domain of TLR4,    -   wherein “eRAGE” is the extracellular domain of RAGE,    -   wherein “Fc” is the immunoglobulin Fc region, and    -   wherein “−” is a peptide linker or a peptide bond.

In some embodiments, the fusion protein comprises a formula selectedfrom the group consisting of:

-   -   eCD33-Fc-Avi,    -   eTLR4-Fc-Avi,    -   eRAGE-Fc-Avi,    -   eCD33-eTLR4-Fc-Avi,    -   eCD33-eRAGE-Fc-Avi,    -   eTLR4-eRAGE-Fc-Avi,    -   eRAGE-eTLR4-Fc-Avi,    -   eCD33-eTLR4-eRAGE-Fc-Avi,    -   eCD33-eRAGE-eTLR4-Fc-Avi,    -   eTLR4-eCD33-eRAGE-Fc-Avi,    -   eRAGE-eCD33-eTLR4-Fc-Avi,    -   eTLR4-eRAGE-eCD33-Fc-Avi, and    -   eRAGE-eTLR4-eCD33-Fc-Avi    -   wherein “eCD33” is the extracellular domain of human CD33,    -   wherein “eTLR4” is the extracellular domain of TLR4,    -   wherein “eRAGE” is the extracellular domain of RAGE,    -   wherein “Fe” is the immunoglobulin Fe region,    -   wherein “Avi” is an optional biotin acceptor peptide that can be        biotinylated with biotin ligase (BirA) in the presence of biotin        and ATP, and    -   wherein “−” is a peptide linker or a peptide bond.

The S100A9 protein can be present as a monomer or dimer. For example,the S100A9 protein can be in association with S100A8 protein asheterodimer. In these cases, the CD33/S100A9 antagonist binds andsequesters the S100A8/A9 heterodimer complex.

In some cases, the CD33/S100A9 antagonist is multivalent, e.g., itcontains at least 2, 3, or more S100A9-binding domains. For example, theCD33/S100A9 antagonist can be a chimeric fusion protein comprising acombination of one or more CD33 ectodomains, one or more TLR4ectodomains, and/or one or more RAGE ectodomains. The fusion protein canfurther comprise an immunoglobulin heavy chain constant region (Fc),e.g., from IgG4, IgG2 or IgG1.

Multiple copies of the fusion protein can also be combined to form amultivalent complex. Therefore, in some embodiments, two, three, four,five, or more fusion proteins can be linked to a core molecule orparticle. For example, the Fc portion can be biotinylated. Thebiotinylated fusion protein can then be conjugated to a multimeric(e.g., tetrameric) streptavidin. In other embodiments, two or morefusion proteins are conjugated to the surface of a liposome or othermicroparticle. The multivalent complex can be homogeneous, i.e.,containing multiple copies of one type of one fusion protein, or it canbe heterogeneous. For example, the multivalent complex can containcombinations of CD33, TLR4, and RAGE fusion proteins. In addition, themultivalent complex can contain multivalent fusion proteins, i.e., atleast two fusion protein containing two or more S100A9-binding domains.

In other embodiments, the CD33/S100A9 inhibitor binds and inhibitsendogenous CD33 receptor on MDSCs. Therefore, in some embodiments, theCD33/S100A9 antagonist is a molecule containing a CD33 binding domain.For example, the CD33/S100A9 inhibitor can be a recombinant protein thatbinds CD33 without activating MDSCs and competes for binding ofendogenous S100A9. For example, the CD33/S100A9 inhibitor can be amutant or truncated variant of S100A9, i.e., dominant negative S100A9.

In some embodiments, the CD33/S100A9 inhibitor is an antibody or aptamerthat specifically binds CD33 or S100A9 thereby inhibiting endogenousCD33/S100A9 activation.

Also disclosed are methods for treating a disease or condition in asubject that is caused or exacerbated by S100A9 activity, comprisingadministering to the subject a composition comprising a CD33/S100A9antagonist disclosed herein.

Also disclosed are methods for identifying an agent for treating MDS. Insome embodiments, the methods involve screening candidate agents for theability to prevent S100A9 binding to CD33. In other embodiments, themethods involve screening candidate agents for the ability to preventactivation of CD33 by S100A9.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIGS. 1A to 1N show increased accumulation and function of MDSC in BMcells from MDS patients. FIG. 1A shows percent of MDSCs in the BM-MNCsof MDS (n=12), age-matched healthy (n=8) and non-MDS cancer specimens(n=8, 4 breast 4 lymphoma, P<0.0001). FIG. 1B shows chromosome 7 FISH ofsorted MDSC or non-MDSC from MDS BM-MNC (n=5, CEP7 and 7q31). FIGS. 1Cand 1D show ³H-thymidine incorporation (FIG. 1C) and IFN-γ ELISA (FIG.1D) of stimulated autologous T cells co-cultured with sorted MDS-MDSCsat 1:0.25 and 1:0.5 ratios (T cells:MDSC). Error bars denote standarddeviation of three separate patient samples tested in triplicate. FIG.1E shows BrDu incorporation of stimulated T cells after admixing withautologous unsorted, MDSCs depleted (−MDSC) or remixed (+MDSC) BM-MNCs.FIGS. 1F to 1I show sorted MDSCs from MDS or healthy donor BM tested forIL-10 (FIG. 1F), TGFβ (FIG. 1G), arginase (FIG. 1H) and NO (FIG. 1I)production by ELISA after 24 hours of culture. FIG. 1J showsMDSC:erythroid precursor contact zone of admixed sorted MDS-MDSC andautologous erythroid precursors at a ratio of 1:3 (MDSC:erythroidprecursor) by microscopy at 0 and 30 minutes. Cells were stained forCD71, glycophorin A, CD33 and granzyme B. FIG. 1K shows sorted MDSCsfrom MDS or healthy donors labeled with CD33 and granzyme B co-incubatedwith purified autologous erythroid precursors (0 or 30 min) andmonitored by microscopy. (L) Counts of MDSC-HPC conjugate mobilizedgranules. FIG. 1M shows Annexin V exposure on erythroid precursors(CD71⁺CD235a⁺) incubated with or without sorted autologous MDS-BM MDSC.FIG. 1N shows colony forming ability of unsorted, −MDSC or remixed(+MDSC) MDS BM-MNCs (ratio of 1:3, *, P<0.005, **, P<0.001).

FIGS. 2A to 2G show CD33 signals to enhance MDSC suppressive functions.FIG. 2A shows BM-MNCs from MDS patients (n=12), age matched healthydonors (n=8) and non-MDS cancers (n=8) analyzed for CD33's Meanfluorescence intensity (MFI), *=P<0.0005. FIG. 2B shows concentration ofIL-10, TGF-β, and VEGF from the supernatant of CD33 (or isotype)cross-linked U937 cells. Bars represent mean±SEM of three wells on threeseparate experiments. FIG. 2C shows BM-MNCs isolated from healthy donorsand infected with an adenoviral vector containing either GFP (Ad-GFP) orCD33 (Ad-CD33) for 72 hours before flow cytometric analysis of themature myeloid markers, CD11c, CD80, and CCR7, with non-infected cellsas a control. FIG. 2D shows results of colony formation assay whereafter sorting out MDSCs from the BM of MDS patients, the remaining MDSCnegative cells were cultured with MDSCs that have been mock infected orinfected with lentiviral vector (LV) containing non-targeted shRNA orCD33 shRNA (shCD33) for 14 days. The MDSCs were also cultured for 72hours after infection with LV containing constructs described abovebefore culturing with MDSC negative BM cells. *, P<0.001, **, P<0.0001,versus cells treated with control shRNA. FIG. 2E to 2G shows IL-10 (FIG.2E) and TGF-β (FIG. 2F) in the supernatants assayed by ELISA. *, P<0.05,versus cells treated with control shRNA. FIG. 2G shows arginase activityin the shCD33 treated cells. *P<0.05, versus cells treated with controlshRNA.

FIGS. 3A to 3O show identification of S100A9 as a native ligand forCD33. FIG. 3A shows Coomasie blue staining of BM lysates precipitatedwith either control IgG or CD33-fusion. FIG. 3B shows transfectedSJCRH30 cells (S100A8 on top left and S100A9 lower left) stained withCD33-fusion (APC). FIG. 3C shows S100A9 capture ELISA of lysates fromun-transfected (negative) or S100A9 transfected cells. Secondaryantibody was either anti-S100A9 (positive control) or CD33-fusion. FIG.3D shows serial dilution of both AD293 and SJCRH30 cell lysates, eitherun-transfected or transfected with vector or S100A9, onto a PVDFmembrane and blotted with CD33-fusion. Coomasie blue staining serves asloading control. FIG. 3E shows S100A9 immunoprecipitation of SJCRH30CD33/S100A9 co-transfected cell lysate blotted against CD33. FIG. 3Fshows PBMC and BM-MNCs from healthy and MDS samples immunoprecipitatedwith CD33-fusion and blotted for S100A9. FIG. 3G showsimmunofluorescence staining of recombinant human S100A9-DDK incubatedwith either CD33-transfected (top panel) or vector-transfected (lowerpanel) SJCRH30 cells at indicated time points. DAPI=nuclei,APC-DDK=rhS100A9. FIGS. 3H to 3I show treatment of SJCRH30-CD33 cellswith rhS100A9 induced IL-10 (FIG. 3H) and TGF-β expression (FIG. 3I).FIGS. 3J to 3K show treatment of U937 cells (high CD33 expressionmyeloid cell line) with rhS100A9 also induces IL-10 (FIG. 3J) and TGFβexpression (FIG. 3K). FIG. 3L shows S100A9 protein concentration in theplasma of MDS patients (n=6) measured by ELISA. FIGS. 3M to 3N showMDS-MDCS treated with 1 μg of rhS100A9 stained for CD33-FITC andanti-DDK-APC (FIG. 3M) and immunoprecipitated with anti-CD33 antibodyfollowed by blotting with anti-SHP-1 (FIG. 3N). FIG. 3O shows BM plasmafrom either healthy donors (n=3) or MDS-patients (n=3) used to assaySHP-1 recruitment. In all experiments, error bars represent the SEM ofthree separate experiments.

FIGS. 4A to 4N show S100A9 signaling through CD33 in MDS BM isassociated with MDSC activation and suppressive function. FIGS. 4A to 4Hshow healthy BM cells infected with adenovirus containing GFP or CD33expression vectors assessed by Q-PCR for the expression of IL-10 (FIG.4A), TGF-β (FIG. 4C), ARG2 (FIG. 4E) or NOS2 (FIG. 4F), or by ELISA forIL-10 (FIG. 4B) and TGF-β (FIG. 4D). Q-PCR (FIG. 4G) and flow cytometryof GFP expression (FIG. 4H) determined transfection efficiency. FIGS. 4Iand 4J show healthy BM cells' RAGE, TLR4, CD33 or their combinationblocked prior to culturing cells by themselves or with lug of S100A9 for48 hours to determine IL-10 gene and protein expression (Q-PCR andELISA, (FIG. 4I)) or TGF-β gene and protein expression (FIG. 4J). FIG.4K shows silencing S100A8 and S100A9 expression in primary MDS-BM cellsusing specific shRNA (demonstrated by western blot). FIGS. 4L to 4N showthat silencing inhibits the expression of IL-10 (FIG. 4L) and TGF-β(FIG. 4M). *, P<0.01, **, P<0.001, versus cells treated with controlshRNA. FIG. 4N shows blocking S100A8 and S100A9 expression by specificshRNA promotes colony formation in BM cells isolated from patients withMDS, *, P<0.05, versus cells treated with control shRNA. In allexperiments, error bars represent the SEM of triplicate determinationwith three separate primary specimens.

FIGS. 5A to 5P show S100A9Tg mice have increased accumulation andactivation of MDSC and display dysplastic features that recapitulatehuman MDS pathology. FIG. 5A shows Gr1⁺CD11b⁺ MDSC accumulation inBM-MNCs isolated from S100A9Tg mice at 6, 18 or 24 weeks, S100A9KO or WTmice at both 6 and 24 weeks. FIG. 5B shows percent of MDSC from BM,spleen and PBMC of S100A9Tg mice at 6, 18 and 24 weeks of age by flowcytometry. FIGS. 5C to 5D show spleen cells assayed against thematuration markers F4/80⁺Gr1⁻ (FIG. 5C) and I-Ad⁺ (FIG. 5D). FIG. 5Eshows FACS sorted Gr-1⁺CD11b⁺ cells (+MDSC) from the BM of micedescribed in FIG. 5A remixed back with autologous 1×10⁵ MDSC-negativepopulation (containing HSPC, −MDSC) at 1:1 ratio for 14 days beforeevaluating colony formation. An MDSC-negative population was used as thecontrol. FIG. 5F shows MDSCs from WT, S100A9Tg and S100A9 KO mice FACSsorted and incubated in a 96-well plate for 24 hrs after which IL-10 andTGF-β production were measured by ELISA. FIGS. 5G to 5N show comparisonof the hematopathological analysis of WT (FIGS. 5G-5J) and S100A9Tg mice(FIGS. 5K-5N). MDS-BM primary specimens were tested for the location ofS100A9 in CD33 positive cells (FIG. 5O) and CD34 positive cells (FIG.5P). Flow figures representative of triplicate experiments.

FIGS. 6A to 6I show mix transplant of S100A9 enriched HSC and WT HSCcontinues effects on hematopoiesis. FIG. 6A shows proportion of MDSC inmice lethally irradiated mice (900Gy) transplanted with enriched HSCsfrom either WT, S100A9Tg or a 1:1 mixture of the two at 8 weeks(post-engraftment). Figure representative of 5 transplant experiments.FIG. 6B shows GFP expression of MDSCs in FIG. 6A. FIG. 6C shows percentof LSK HSC, defined as Lineage⁻cKit*Sca-1⁺, in lethally irradiated miceafter transplant with WT, S100A9Tg or 1:1 mix of enriched HSCs. FIGS. 6Dto 6F show proportion and concentration of white blood cells (WBC) (FIG.6D), hemoglobin (HGB) (FIG. 6E) and RBCs (FIG. 6F) measured weekly byCBC post-transplant. Error bars are the SEM of n=5. FIG. 6G showspercentage of CD34 positive cells in MDSC-depleted MDS-BM specimenstreated with or without S100A9 for 48 hours. FIG. 6H shows sameexperiment as in FIG. 6G, assessing surface expression of Annexin V andPI after treatment with S100A9. FIG. 6I shows healthy human CD34 cells(Lonza Wakersfield) were treated as in FIG. 6H and cultured for 48 hoursfollowed by AnnexinV/PI flow cytometric analysis.

FIGS. 7A to 7I show targeting MDSC activation and signaling can improvesuppressive BM microenvironment. FIGS. 7A to 7B show ATRA decreasesMDSCs in the BM of S100A9Tg mice (FIG. 7A) and promotes the expressionof myeloid maturation markers (FIG. 7B). FIG. 7C shows BFU colonyformation of WT and S100A9Tg mice BM cells treated with ATRA. All of thecultures were duplicates. *, P<0.05, between ATRA treated and vehicletreated S100A9Tg mice.

FIG. 7D shows the number of RBC, WBC and platelets from CBC analysis ofATRA treated and untreated mice. *, P<0.05, between ATRA treated andvehicle treated S100A9Tg mice. FIG. 7E shows relative expression levelsof DAP12 from isolated MDSC from either healthy or MDS specimens by qPCR(n=5). FIG. 7F shows AD293 cells transfected with either vector,WT-DAP12, dominant negative DAP12 (DN) or active DAP12 (P23) for 48hours and analyzed by western blot for the expression of phosphorylatedor total Syk and ERK. This is representative of three independentexperiments. MDSC were isolated from the BM of MDS patients and infectedwith adenoviral vector containing either WT or active DAP12 (P23) for 48or 72 hours. FIGS. 7G and 7H show surface expression of CD14 or CD15(FIG. 7G) or the maturation markers CD80, CCR7 and CD11c (FIG. 7H)analyzed by flow cytometry. FIG. 7I shows MDSCs purified from BM-MNCs ofMDS patients by FACS sorting and cells infected with LV-WT DAP12 orLV-P23. Colony formation assays were performed in methylcellulose for 14days. Results are shown as mean±SEM of 7 patients. *, P<0.01, **,P<0.001, versus cells infected with LV-WT.

FIG. 8 shows percent engraftment in transplanted mice. Wild type femaleFVB/NJ mice were transplanted with enriched HSC from either WT, S100A9Tgor a 1:1 ratio of WT:Tg male cells. The percent of engraftment wasassessed by measuring the expression of the SRY gene by qPCR normalizedagainst un-transplanted male mice BM cells. GAPDH serves as the internalcontrol.

FIG. 9 is a model of CD33/Siglec 3-mediated signaling either throughITIM or ITAM motifs. CD33 has two cytosolic ITIM motifs. Src kinasesphosphorylate tyrosine residues present in the ITIMS after CD33cross-linking or upon interaction with its ligand(s). PhosphorylatedITIMs recruit and activate phosphatases (SHP1, SHP2, or SHIP-1),resulting in down regulation of MAPK and ultimately, cellular inhibitionand the production of inhibitory cytokines. Activation of CD33 alsoinduces S100A8 and S100A9 expression, which are secreted and act asheterodimers for CD33 or TLR4, and as such, mediates inflammation andMDSC activation. Siglecs that lack ITIMs possess charged amino acids intheir trans-membrane domain, which allow association with DAP12, anITAM-bearing activating adaptor. As is the case with ITIMs, Src kinasesalso phosphorylate tyrosine residues in the ITAM of DAP12. Syk kinaseand ZAP70 are then recruited and perpetuate downstream cellularactivation, differentiation, and/or maturation.

FIG. 10 shows t effect of active DAP12 on immature DC maturation.Primary DCs were prepared from healthy donors and infected withadenoviral vectors containing GFP alone, WT-DAP12, dnDAP12 and activeDAP12 (Ad-P23) as indicated. The cells were cultured for 72 hrs beforeflow cytometric analysis using mature DC surface marker as indicated.Mock infected DC and Isotype IgG included in control group. Eachexperimental construct was compared to the empty-vector control (filledhistograms), and infected cells were gated on GFP prior to analysis.

FIG. 11 illustrates exemplary embodiments of a CD33/S100A9 antagonist.FIGS. 11A to 11G show a fusion protein contain the Fc portion of IgG,and a biotin acceptor peptide (e.g., AviTag™ sequence) for biotinylationby BirA or antibody recognition. The fusion protein in FIGS. 11A, 11C,and 11G contain the ectodomain of CD33. The fusion protein of 11B and11D contains only the variable region of the ectodomain of CD33(“vCD33”). The vCD33 region in FIGS. 11C and 11D contains singlenucleotide polymorphisms (“SNPs”) compared to the vCD33 in FIGS. 11A and11B. The fusion protein depected in FIGS. 11E, 11F, and 11G contain theectodomain of Toll like receptor 4 (TLR4). The fusion proteins in FIGS.11F and 11G also contain the ectodomain of CD33 (FIG. 11G) or only thevCD33 region (FIG. 11F). FIG. 11H shows a multimeric complex formed bythe biotinylation of the AviTag™ sequence and the conjugation of thesefusion proteins to a tetrameric streptavidin (“SA”). FIG. 11I shows amultimeric complex formed by the incorporation of antibodies thatspecifically bind AviTag™ into a liposome.

FIGS. 12A and 12B show fold change of active caspase-1 and IL-βgeneration normalized to plasma treated control in four cellpopulations. Active caspase-1 and IL-β generation were assessed in fourpopulations by flow cytometry after treatment with CD33-IgG: stem cells(CD34+CD38−), progenitors (CD34+CD38+), erythroids (CD71+), and myeloidcells (CD33+). Fold change of active caspase-1 MFI (FIG. 12A) and IL-βgeneration (FIG. 12B).

FIGS. 13A and 13B show fold change of active caspase-1 and IL-βgeneration normalized to plasma treated control in four cellpopulations. Active caspase-1 and IL-1 generation were assessed in fourpopulations by flow cytometry after treatment with a related pathwayinhibitor: stem cells (CD34+CD38−), progenitors (CD34+CD38+), erythroids(CD71+), and myeloid cells (CD33+). Fold change of active caspase-1 MFI(FIG. 13A) and IL-β generation (FIG. 13B).

FIGS. 14A to 14C show neutralization of plasma S100A9 by CD33 Chimeratrap enhances colony forming capacity in MDS patient specimens. FIGS.14A to 14C show erythroid burst-forming units (BFU-E) (FIG. 14B),multipotential colony forming units (CFU-GEMM) (FIG. 14A), andgranulocyte/macrophage colony forming units (CFU-GM) (FIG. 14C) in MDSpatient specimens treated with IgG, Plasma, or 0.1, 0.5, or 1.0 μg ofCD33-IgG chimeric trap.

FIGS. 15A to 15C show change in colony forming capacity in four LR-MDSspecimens treated with CD33 chimera. BM-MNC from each patient wereincubated with autologous BM plasma and increasing concentrations ofCD33-IgG, and were plated in four replicates per treatment condition inmethylcellulose. Colonies were counted fourteen days after plating, andwere averaged for each patient. The increase in CFC is represented asthe fold change normalized to plasma-incubated control for GEMM (FIG.15A), erythroid (FIG. 15B), and GM (FIG. 15C) colonies, respectively.

FIGS. 16A to 16E show CD33-chimera trap suppresses pyroptosis-relatedgene expression in MDS BM-MNC. BM-MNC were isolated from five low riskMDS patients and incubated with autologous BM plasma and increasingconcentrations of the CD33 chimera. BM-MNC isolated from five normaldonors were used for comparison. RNA was isolated and qPCR was carriedout on pyroptosis-related genes Capsapse-1 (FIG. 16A), IL-β (FIG. 16B),NLRP1 (FIG. 16C), NLRP3 (FIG. 16D), and IL-18 (FIG. 16E). Geneexpression is represented as the fold change normalized to the normaldonors.

DETAILED DESCRIPTION

Immature myeloid-derived suppressor cells (MDSC), known to accumulate intumor bearing mice and cancer patients, are site-specific inflammatoryand T cell immunosuppressive effector cells that contribute to cancerprogression (Gabrilovich, D. I., et al. 2009. Nat Rev Immunol 9:162-174;Kusmartsev, S., et al. 2006. Cancer Immunol Immunother 55:237-245).Their suppressive activity is in part driven by inflammation-associatedsignaling molecules, such as the danger-associated molecular pattern(DAMP) heterodimer S100A8/S100A9 (also known as myeloid-related protein(MRP)-8 and MRP-14, respectively), through ligation of TLR4 (Ehrchen, J.M., et al. 2009. J Leukoc Biol 86:557-566; Vogl, T., et al. 2007. NatMed 13:1042-1049). Murine CD11b⁺Gr1⁺ MDSCs form the basis of the vastmajority of the mechanistic studies, however, much less has beenreported on their human counterparts. Human MDSCs lack most markers ofmature immune cells (LIN⁻, HLA-DR⁻) but possess CD33, the prototypicalmember of Sialic acid-binding immunoglobulin-like (Ig) super-family oflectins (Siglec) (Gabrilovich, D. I., et al. 2009. Nat Rev Immunol9:162-174; Talmadge, J. E. 2007. Clin Cancer Res 13:5243-5248; Talmadge,J. E., et al. 2007. Cancer Metastasis Rev 26:373-400; Crocker, P. R., etal. 2007. Nat Rev Immunol 7:255-266). Importantly, while its preciseaction is unknown, CD33 possesses an immunoreceptor tyrosine-basedinhibitory motif (ITIM) that is associated with immune suppression(Crocker, P. R., et al. 2007. Nat Rev Immunol 7:255-266).

It is shown herein that LIN⁻HLA-DR⁻CD33⁺ MDSCs specifically accumulatein the BM of MDS patients (herein referred to as MDS-MDSC) and impairhematopoiesis through a mechanism that involves S100A9 as an endogenousligand for CD33 initiated signaling. Importantly, using S100A9transgenic (S100A9Tg) mice, it is shown that sustained activation ofthis inflammatory pathway leads to the development of MDS, and that thishematologic phenotype is rescued by strategies that suppress CD33ITIM-signaling. The disclosed finding that S100A9 ligates CD33 to induceMDSC expansion indicates that targeting this pathway can provide atherapeutic approach for the treatment of MDS. Finally, the discovery ofthis signaling pathway verifies the role of S100A9 as an importantinitiator of immune-suppression. S100A9Tg mice may therefore serve as auseful model for the study of MDS pathogenesis, treatment and theoverall role of MDSC in cancer.

Therefore, disclosed are compositions and methods for treating MDS thatinvolve the use of a CD33/S100A9 inhibitor to inhibit activation ofmyeloid-derived suppressor cells (MDSCs).

CD33/S100A9 Inhibitor

In some embodiments, the CD33/S100A9 inhibitor binds and sequestersendogenous S100A9 and inhibits its binding to CD33 receptor on MDSCs.Therefore, in some embodiments, the CD33/S100A9 antagonist is a moleculecontaining a S100A9 binding domain. In other embodiments, theCD33/S100A9 inhibitor binds and inhibits endogenous CD33 receptor onMDSCs. Therefore, in some embodiments, the CD33/S100A9 antagonist is amolecule containing a CD33 binding domain.

Soluble Receptors

For example, the CD33/S100A9 inhibitor can be a soluble CD33 receptor. A“soluble receptor” is a receptor polypeptide that is not bound to a cellmembrane. Soluble receptors are most commonly ligand-binding receptorpolypeptides that lack transmembrane and cytoplasmic domains.

Soluble receptors can comprise additional amino acid residues, such asaffinity tags that provide for purification of the polypeptide orprovide sites for attachment of the polypeptide to a substrate, orimmunoglobulin constant region sequences. Many cell-surface receptorshave naturally occurring, soluble counterparts that are produced byproteolysis. Soluble receptor polypeptides are said to be substantiallyfree of transmembrane and intracellular polypeptide segments when theylack sufficient portions of these segments to provide membrane anchoringor signal transduction, respectively.

For example, the CD33/S100A9 antagonist can be a chimeric fusion proteincomprising the ectodomain of CD33, Toll like Receptor 4 (TLR4), Receptorfor Advanced Glycation End Products (RAGE), or a combination thereof.This ectodomain of CD33, TLR4, and/or RAGE can be any fragment of CD33,TLR4, and/or RAGE capable of binding S100A9.

Fusion proteins, also known as chimeric proteins, are proteins createdthrough the joining of two or more genes which originally coded forseparate proteins. Translation of this fusion gene results in a singlepolypeptide with function properties derived from each of the originalproteins. Recombinant fusion proteins can be created artificially byrecombinant DNA technology for use in biological research ortherapeutics. Chimeric mutant proteins occur naturally when alarge-scale mutation, typically a chromosomal translocation, creates anovel coding sequence containing parts of the coding sequences from twodifferent genes.

The functionality of fusion proteins is made possible by the fact thatmany protein functional domains are modular. In other words, the linearportion of a polypeptide which corresponds to a given domain, such as atyrosine kinase domain, may be removed from the rest of the proteinwithout destroying its intrinsic enzymatic capability. Thus, any of theherein disclosed functional domains can be used to design a fusionprotein.

A recombinant fusion protein is a protein created through geneticengineering of a fusion gene. This typically involves removing the stopcodon from a cDNA sequence coding for the first protein, then appendingthe cDNA sequence of the second protein in frame through ligation oroverlap extension PCR. That DNA sequence will then be expressed by acell as a single protein. The protein can be engineered to include thefull sequence of both original proteins, or only a portion of either.

If the two entities are proteins, often linker (or “spacer”) peptidesare also added which make it more likely that the proteins foldindependently and behave as expected. Especially in the case where thelinkers enable protein purification, linkers in protein or peptidefusions are sometimes engineered with cleavage sites for proteases orchemical agents which enable the liberation of the two separateproteins. This technique is often used for identification andpurification of proteins, by fusing a GST protein, FLAG peptide, or ahexa-his peptide (aka: a 6xhis-tag) which can be isolated using nickelor cobalt resins (affinity chromatography).

Amino acid sequences for suitable CD33, TLR4, and RAGE ectodomains areknown and adaptable for use in the disclosed compositions and methods.For example, the ectodomain of Homo sapiens Myeloid cell surface antigenCD33 (Uniprot P20138 [aa. 18-259] can have the following amino acidsequence:

18-DPN FWLQVQESVT VQEGLCVLVP CTFFHPIPYY DKNSPVHGYW FREGAIISRD SPVATNKLDQEVQEETQGRF RLLGDPSRNN CSLSIVDARR RDNGSYFFRM ERGSTKYSYK SPQLSVHVTDLTHRPKILIP GTLEPGHSKN LTCSVSWACE QGTPPIFSWL SAAPTSLGPR TTHSSVLIITPRPQDHGTNL TCQVKFAGAG VTTERTIQLN VTYVPQNPTT GIFPGDGSGK QETRAGVVH-259(SEQ ID NO:1), or a fragment or variant thereof, e.g., having an aminoacid sequence having at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ IDNO:1, wherein the fragment or variant is capable of binding S100A9.

There are at least three known single nucleotide polymorphisms (“SNPs”)in the ectodomain of CD33 (i.e., W22R, R69G, S128N). Therefore, theextracellular domain of Homo sapiens CD33 can have the amino acidsequence of SEQ ID NO:1 with any one or more of these SNPs. For example,the extracellular domain of Homo sapiens CD33 can also have thefollowing amino acid sequence:

18-DPN FX₁LQVQESVT VQEGLCVLVP CTFFHPIPYY DKNSPVHGYW FREGAIISX₂DSPVATNKLDQ EVQEETQGRF RLLGDPSRNN CSLSIVDARR RDNGSYFFRM ERGSTKYX₃YKSPQLSVHVTD LTHRPKILIP GTLEPGHSKN LTCSVSWACE QGTPPIFSWL SAAPTSLGPRTTHSSVLIIT PRPQDHGTNL TCQVKFAGAG VTTERTIQLN VTYVPQNPTT GIFPGDGSGKQETRAGVVH-259, where X₁ is W or R; wherein X₂ is R or G; and wherein X₃is S or N (SEQ ID NO:2).

In some cases, the CD33 ectodomain contains only the variable region ofCD33 (“vCD33”). For example, in some embodiments, the vCD33 portion hasthe following amino acid sequence:

19-PN FWLQVQESVT VQEGLCVLVP CTFFHPIPYY DKNSPVHGYW FREGAIISRD SPVATNKLDQEVQEETQGRF RLLGDPSRNN CSLSIVDARR RDNGSYFFRM ERGSTKYSYK SPQLSVHVTD-135(SEQ ID NO:3), or a fragment or variant thereof, e.g., having an aminoacid sequence having at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ IDNO:3, wherein the fragment or variant is capable of binding S100A9. ThevCD33 can also have any one or more of the disclosed SNPs (i.e., W22R,R69G, S128N). Therefore, in some embodiments, the vCD33 portion has thefollowing amino acid sequence:19-PN FX₁LQVQESVT VQEGLCVLVP CTFFHPIPYY DKNSPVHGYW FREGAIISX₂DSPVATNKLDQ EVQEETQGRF RLLGDPSRNN CSLSIVDARR RDNGSYFFRM ERGSTKYX₃YKSPQLSVHVTD-135, where X₁ is W or R; wherein X₂ is R or G; and wherein X₃is S or N (SEQ ID NO:4).

The variable domain of CD33 can be further mutated to create moreglycosylation sites in order to enhance binding to S100A9. Thesemutations can be screened in silico and/or tested in vitro to evaluateS100A9 binding.

N-linked carbohydrates are linked through N-Acetylglucosamine andasparagines. The N-linked consensus sequence is Asn-X₁-X₂, wherein X₁ isany amino acid other than Pro, and X₂ is Ser or Thr. Most O-linkedcarbohydrate covalent attachments involve a linkage between themonosaccharide N-Acetylgalactosamine and the amino acids serine orthreonine.

Non-limiting examples of mutant sites will be but not limited at CD33residues that can be mutated to create more glycosylation sites includeQ26N, P40N, L78T, and E84N. Other residues can be identified and testedusing routine methods. For example, protein-protein reactions can beassayed using a binding affinity assay, such as an assay that usessurface plasmon resonance (SPR) to detect unlabeled interactants in realtime, e.g., using a Biacore™ Sensor Chip (GE Healtchare).

The extracellular domain of Homo sapiens Toll-like receptor 4 (TLR4)(Uniprot 000206 [aa. 24-631]) can have the following amino acidsequence:

24-ESWEPCV EVVPNITYQC MELNFYKIPD NLPFSTKNLD LSFNPLRHLG SYSFFSFPELQVLDLSRCEI QTIEDGAYQS LSHLSTLILT GNPIQSLALG AFSGLSSLQK LVAVETNLASLENFPIGHLK TLKELNVAHN LIQSFKLPEY FSNLTNLEHL DLSSNKIQSI YCTDLRVLHQMPLLNLSLDL SLNPMNFIQP GAFKEIRLHK LTLRNNFDSL NVMKTCIQGL AGLEVHRLVLGEFRNEGNLE KFDKSALEGL CNLTIEEFRL AYLDYYLDDI IDLFNCLTNV SSFSLVSVTIERVKDFSYNF GWQHLELVNC KFGQFPTLKL KSLKRLTFTS NKGGNAFSEV DLPSLEFLDLSRNGLSFKGC CSQSDFGTTS LKYLDLSFNG VITMSSNFLG LEQLEHLDFQ HSNLKQMSEFSVFLSLRNLI YLDISHTHTR VAFNGIFNGL SSLEVLKMAG NSFQENFLPD IFTELRNLTFLDLSQCQLEQ LSPTAFNSLS SLQVLNMSHN NFFSLDTFPY KCLNSLQVLD YSLNHIMTSKKQELQHFPSS LAFLNLTQND FACTCEHQSF LQWIKDQRQL LVEVERMECA TPSDKQGMPVLSLNITCQMN K-631 (SEQ ID NO:5), or a fragment or variant thereof, e.g.,having an amino acid sequence having at least 65%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequenceidentity to SEQ ID NO:5, wherein the fragment or variant is capable ofbinding S100A9.

The ectodomain of TLR4 has previously been used in a fusion protein withmyeloid differentiation factor 2 (MD-2) to function as alipopolysaccharide (LPS) trap. MD-2 is necessary for TLR4 to bind LPS.However, it is not necessary for TLR4 to bind and trap S100A9.Therefore, in some embodiments, the fusion protein containing TLR4 doesnot also contain MD-2. In other embodiments, the fusion protein containsboth TLR4 and CD33 ectodomains.

The extracellular domain of Homo sapiens Receptor for Advanced GlycationEnd Products (RAGE) (Accession No. NP_001127 [aa. 23-342]) can have thefollowing amino acid sequence:

23-AQNITARI GEPLVLKCKG APKKPPQRLE WKLNTGRTEA WKVLSPQGGG PWDSVARVLPNGSLFLPAVG IQDEGIFRCQ AMNRNGKETK SNYRVRVYQI PGKPEIVDSA SELTAGVPNKVGTCVSEGSY PAGTLSWHLD GKPLVPNEKG VSVKEQTRRH PETGLFTLQS ELMVTPARGGDPRPTFSCSF SPGLPRHRAL RTAPIQPRVW EPVPLEEVQL VVEPEGGAVA PGGTVTLTCEVPAQPSPQIH WMKDGVPLPL PPSPVLILPE IGPQDQGTYS CVATHSSHGP QESRAVSISIIEPGEEGPTA GSVGGSGLGT LA-342 (SEQ ID NO:6), or a fragment or variantthereof, e.g., having an amino acid sequence having at least 65%, 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% sequence identity to SEQ ID NO:6, wherein the fragment or variant iscapable of binding S100A9.

A receptor extracellular domain of CD33, TLR4, and/or RAGE can beexpressed as a fusion with immunoglobulin heavy chain constant regions,typically an Fc fragment, which contains two constant region domains andlacks the variable region. Such fusions can be secreted as multimericmolecules wherein the Fc portions are disulfide bonded to each other andtwo receptor polypeptides are arrayed in close proximity to each other.This chimeric molecule can be produced as fusion protein, which can beformed by the chemical coupling of the constituent polypeptides or itcan be expressed as a single polypeptide from nucleic acid sequenceencoding the single contiguous fusion protein. A single chain fusionprotein is a fusion protein having a single contiguous polypeptidebackbone. Fusion proteins can be prepared using conventional techniquesin molecular biology to join the two genes in frame into a singlenucleic acid, and then expressing the nucleic acid in an appropriatehost cell under conditions in which the fusion protein is produced. Inparticular, the CD33/S100A9 inhibitor can be a chimeric fusion proteincomprising the ectodomain of CD33, TLR4, and/or RAGE and animmunoglobulin heavy chain constant region (Fc), e.g., from IgG1.

In some cases, the CD33/S100A9 antagonist is multivalent, e.g., itcontains at least 2, 3, or more S100A9-binding domains. In some cases,the antagonist contains at least one CD33 ectodomain and aS100A9-binding domain selected from the group consisting of a TLR4ectodomain and a RAGE ectodomain.

Multiple copies of the fusion protein can also be combined to form amultivalent complex. Therefore, in some embodiments, two or more fusionproteins can be linked to a core molecule or particle. In its simplestform, a multivalent complex comprises a multimer of two or three or fouror more of the disclosed fusion proteins associated (e.g. covalently orotherwise linked) with one another preferably via a linker molecule.Suitable linker molecules include multivalent attachment molecules suchas avidin, streptavidin and extravidin, each of which can have fourbinding sites for biotin.

In some embodiments, the fusion protein can be biotinylated. Oncebiotinylated, the biotinylated fusion protein can then be conjugated toa tetrameric streptavidin. Proteins can be biotinylated chemically orenzymatically. Chemical biotinylation utilises various conjugationchemistries to yield nonspecific biotinylation of amines, carboxylates,sulfhydryls and carbohydrates. Enzymatic biotinylation results inbiotinylation of a specific lysine within a certain sequence by abacterial biotin ligase. Most chemical biotinylation reagents consist ofa reactive group attached via a linker to the valeric acid side chain ofbiotin. This linker can also mediate the solubility of biotinylationreagents; linkers that incorporate poly(ethylene) glycol (PEG) can makewater-insoluble reagents soluble or increase the solubility ofbiotinylation reagents that are already soluble to some extent.

Enzymatic biotinylation is most often carried out by genetically linkingthe protein of interest at its N-terminus, C-terminus or at an internalloop to a 15 amino acid biotin acceptor peptide, also termed AviTag™ orAcceptor Peptide (AP). The tagged protein is then incubated with biotinligase (BirA) in the presence of biotin and ATP. Enzymatic biotinylationcan be carried out in vitro but BirA also reacts specifically with itstarget peptide inside mammalian and bacterial cells and at the cellsurface, while other cellular proteins are not modified. Therefore, thefusion protein can further contain a biotin acceptor peptide that can bebiotinylated with BirA in the presence of biotin and ATP. For example,the Fc portion can be enzymatically biotinylated using the AviTag™system (Avidity, LLC, Aurora, Colorado), which involves incorporating a15 amino acid peptide sequence into the chimeric fusion protein that canbe biotinylated by the BirA enzyme of E. coli. In some embodiments, thisbiotin acceptor peptide has the amino acid sequence GLNDIFEAQKIEWHE (SEQID NO:7).

Oligonucleotides are readily biotinylated in the course ofoligonucleotide synthesis by the phosphoramidite method using biotinphosphoramidite. Upon the standard deprotection, the conjugates obtainedcan be purified using reverse-phase or anion-exchange HPLC.

In other embodiments, two or more fusion proteins are conjugated to thesurface of a liposome or other microparticle to form the multivalentcomplex. A number of reports describe the attachment of antibodies toliposomes. For example, U.S. Pat. No. 5,620,689 discloses so-called“immunoliposomes” in which antibody or antibody fragments effective tobind to a chosen antigen on a B lymphocyte or a T lymphocyte, areattached to the distal ends of the membrane lipids in liposomes having asurface coating of polyethylene glycol chains. In some embodiments, thedisclosed multivalent complexes comprising immunoliposomes containingantibodies that specifically bind the disclosed fusion proteins. Forexample, the antibodies can bind the Fc portion of the fusion protein,or tag on the protein, such as the biotin acceptor peptide.

The multivalent complex can be homogeneous, i.e., containing multiplecopies of one type of one fusion protein, or it can be heterogeneous.For example, the multivalent complex can contain combinations of CD33and TLR4 fusion proteins. In addition, the multivalent complex cancontain multivalent fusion proteins, i.e., at least two fusion proteincontaining two or more S100A9-binding domains.

The disclosed chimeric fusion proteins can also contain a peptide linkersequences connecting the one or more CD33 and/or TLR4 ectodomains toeach other, to the Fc portion, or any combination thereof. For example,the peptide linker can have the amino acid sequence DIEGRMD (SEQ IDNO:8).

In other embodiments, the CD33/S100A9 inhibitor binds and inhibitsendogenous CD33 receptor on MDSCs. For example, the CD33/S100A9inhibitor can be a recombinant protein that binds CD33 withoutactivating MDSCs and competes for binding of endogenous S100A9 (e.g., amutant or truncated variant of S100A9).

Antibodies

Antibodies that can be used in the disclosed compositions and methodsinclude whole immunoglobulin (i.e., an intact antibody) of any class,fragments thereof, and synthetic proteins containing at least theantigen binding variable domain of an antibody. The variable domainsdiffer in sequence among antibodies and are used in the binding andspecificity of each particular antibody for its particular antigen.However, the variability is not usually evenly distributed through thevariable domains of antibodies. It is typically concentrated in threesegments called complementarity determining regions (CDRs) orhypervariable regions both in the light chain and the heavy chainvariable domains. The more highly conserved portions of the variabledomains are called the framework (FR). The variable domains of nativeheavy and light chains each comprise four FR regions, largely adopting abeta-sheet configuration, connected by three CDRs, which form loopsconnecting, and in some cases forming part of, the beta-sheet structure.The CDRs in each chain are held together in close proximity by the FRregions and, with the CDRs from the other chain, contribute to theformation of the antigen binding site of antibodies.

Also disclosed are fragments of antibodies which have bioactivity. Thefragments, whether attached to other sequences or not, includeinsertions, deletions, substitutions, or other selected modifications ofparticular regions or specific amino acids residues, provided theactivity of the fragment is not significantly altered or impairedcompared to the nonmodified antibody or antibody fragment.

Techniques can also be adapted for the production of single-chainantibodies specific to an antigenic protein of the present disclosure.Methods for the production of single-chain antibodies are well known tothose of skill in the art. A single chain antibody can be created byfusing together the variable domains of the heavy and light chains usinga short peptide linker, thereby reconstituting an antigen binding siteon a single molecule. Single-chain antibody variable fragments (scFvs)in which the C-terminus of one variable domain is tethered to theN-terminus of the other variable domain via a 15 to 25 amino acidpeptide or linker have been developed without significantly disruptingantigen binding or specificity of the binding. The linker is chosen topermit the heavy chain and light chain to bind together in their properconformational orientation.

Divalent single-chain variable fragments (di-scFvs) can be engineered bylinking two scFvs. This can be done by producing a single peptide chainwith two VH and two VL regions, yielding tandem scFvs. ScFvs can also bedesigned with linker peptides that are too short for the two variableregions to fold together (about five amino acids), forcing scFvs todimerize. This type is known as diabodies. Diabodies have been shown tohave dissociation constants up to 40-fold lower than correspondingscFvs, meaning that they have a much higher affinity to their target.Still shorter linkers (one or two amino acids) lead to the formation oftrimers (triabodies or tribodies). Tetrabodies have also been produced.They exhibit an even higher affinity to their targets than diabodies.

Aptamers

The term “aptamer” refers to oligonucleic acid or peptide molecules thatbind to a specific target molecule. These molecules are generallyselected from a random sequence pool. The selected aptamers are capableof adapting unique tertiary structures and recognizing target moleculeswith high affinity and specificity. A “nucleic acid aptamer” is a DNA orRNA oligonucleic acid that binds to a target molecule via itsconformation, and thereby inhibits or suppresses functions of suchmolecule. A nucleic acid aptamer may be constituted by DNA, RNA, or acombination thereof. A “peptide aptamer” is a combinatorial proteinmolecule with a variable peptide sequence inserted within a constantscaffold protein. Identification of peptide aptamers is typicallyperformed under stringent yeast dihybrid conditions, which enhances theprobability for the selected peptide aptamers to be stably expressed andcorrectly folded in an intracellular context.

Nucleic acid aptamers are typically oligonucleotides ranging from 15-50bases in length that fold into defined secondary and tertiarystructures, such as stem-loops or G-quartets. Nucleic acid aptamerspreferably bind the target molecule with a K_(d) less than 10⁻⁶, 10,10⁻¹⁰, or 10⁻¹². Nucleic acid aptamers can also bind the target moleculewith a very high degree of specificity. It is preferred that the nucleicacid aptamers have a K_(d) with the target molecule at least 10, 100,1000, 10,000, or 100,000 fold lower than the K_(d) of other non-targetedmolecules.

Nucleic acid aptamers are typically isolated from complex libraries ofsynthetic oligonucleotides by an iterative process of adsorption,recovery and reamplification. For example, nucleic acid aptamers may beprepared using the SELEX (Systematic Evolution of Ligands by ExponentialEnrichment) method. The SELEX method involves selecting an RNA moleculebound to a target molecule from an RNA pool composed of RNA moleculeseach having random sequence regions and primer-binding regions at bothends thereof, amplifying the recovered RNA molecule via RT-PCR,performing transcription using the obtained cDNA molecule as a template,and using the resultant as an RNA pool for the subsequent procedure.Such procedure is repeated several times to several tens of times toselect RNA with a stronger ability to bind to a target molecule. Thebase sequence lengths of the random sequence region and the primerbinding region are not particularly limited. In general, the randomsequence region contains about 20 to 80 bases and the primer bindingregion contains about 15 to 40 bases. Specificity to a target moleculemay be enhanced by prospectively mixing molecules similar to the targetmolecule with RNA pools and using a pool containing RNA molecules thatdid not bind to the molecule of interest. An RNA molecule that wasobtained as a final product by such technique is used as an RNA aptamer.An aptamer database containing comprehensive sequence information onaptamers and unnatural ribozymes that have been generated by in vitroselection methods is available at aptamer.icmb.utexas.edu.

A nucleic acid aptamer generally has higher specificity and affinity toa target molecule than an antibody. Accordingly, a nucleic acid aptamercan specifically, directly, and firmly bind to a target molecule. Sincethe number of target amino acid residues necessary for binding may besmaller than that of an antibody, for example, a nucleic acid aptamer issuperior to an antibody, when selective suppression of functions of agiven protein among highly homologous proteins is intended.

Non-modified nucleic acid aptamers are cleared rapidly from thebloodstream, with a half-life of minutes to hours, mainly due tonuclease degradation and clearance from the body by the kidneys, aresult of the aptamer's inherently low molecular weight. This rapidclearance can be an advantage in applications such as in vivo diagnosticimaging. However, several modifications, such as 2′-fluorine-substitutedpyrimidines, polyethylene glycol (PEG) linkage, etc. are available toincrease the serum half-life of aptamers to the day or even week timescale.

Another approach to increase the nuclease resistance of aptamers is touse a Spiegelmer. Spiegelmers are ribonucleic acid (RNA)-like moleculesbuilt from the unnatural L-ribonucleotides. Spiegelmers are thereforethe stereochemical mirror images (enantiomers) of naturaloligonucleotides. Like other aptamers, Spiegelmers are able to bindtarget molecules such as proteins. The affinity of Spiegelmers to theirtarget molecules often lies in the pico-to nanomolar range and is thuscomparable to antibodies. In contrast to other aptamers, Spiegelmershave high stability in blood serum since they are less susceptible to becleaved hydrolytically by enzymes. Nonetheless, they are excreted by thekidneys in a short time due to their low molar mass. Unlike otheraptamers, Spiegelmers may not be directly produced by the SELEX method.This is because L-nucleic acids are not amenable to enzymatic methods,such as polymerase chain reaction. Instead, the sequence of a naturalaptamer identified by the SELEX method is determined and then used inthe artificial synthesis of the mirror image of the natural aptamer.

Peptide aptamers are proteins that are designed to interfere with otherprotein interactions inside cells. They consist of a variable peptideloop attached at both ends to a scaffold. This double structuralconstraint greatly increases the binding affinity of the peptide aptamerto levels comparable to an antibody.

The variable loop length is typically composed of about ten to twentyamino acids, and the scaffold may be any protein which has goodsolubility. Currently, the bacterial protein Thioredoxin-A is the mostused scaffold protein, the variable loop being inserted within thereducing active site, the two Cysteines lateral chains being able toform a disulfide bridge.

Peptide aptamer selection can be made using different systems, but themost used is currently the yeast two-hybrid system. Peptide aptamer canalso be selected from combinatorial peptide libraries constructed byphage display and other surface display technologies such as mRNAdisplay, ribosome display, bacterial display and yeast display. Theseexperimental procedures are also known as biopannings. Among peptidesobtained from biopannings, mimotopes can be considered as a kind ofpeptide aptamers. All the peptides panned from combinatorial peptidelibraries have been stored in a special database with the name MimoDB.

Pharmaceutical Composition

The disclosed compositions can be used therapeutically in combinationwith a pharmaceutically acceptable carrier. By “pharmaceuticallyacceptable” is meant a material that is not biologically or otherwiseundesirable, i.e., the material may be administered to a subject, alongwith the nucleic acid or vector, without causing any undesirablebiological effects or interacting in a deleterious manner with any ofthe other components of the pharmaceutical composition in which it iscontained. The carrier would naturally be selected to minimize anydegradation of the active ingredient and to minimize any adverse sideeffects in the subject, as would be well known to one of skill in theart.

Suitable carriers and their formulations are described in Remington: TheScience and Practice of Pharmacy (19th ed.) ed. A. R. Gennaro, MackPublishing Company, Easton, PA 1995. Typically, an appropriate amount ofa pharmaceutically-acceptable salt is used in the formulation to renderthe formulation isotonic. Examples of the pharmaceutically-acceptablecarrier include, but are not limited to, saline, Ringer's solution anddextrose solution. The pH of the solution is preferably from about 5 toabout 8, and more preferably from about 7 to about 7.5. Further carriersinclude sustained release preparations such as semipermeable matrices ofsolid hydrophobic polymers containing the antibody, which matrices arein the form of shaped articles, e.g., films, liposomes ormicroparticles. It will be apparent to those persons skilled in the artthat certain carriers may be more preferable depending upon, forinstance, the route of administration and concentration of compositionbeing administered.

Pharmaceutical carriers are known to those skilled in the art. Thesemost typically would be standard carriers for administration of drugs tohumans, including solutions such as sterile water, saline, and bufferedsolutions at physiological pH. The compositions can be administeredintramuscularly or subcutaneously. Other compounds will be administeredaccording to standard procedures used by those skilled in the art.

Pharmaceutical compositions may include carriers, thickeners, diluents,buffers, preservatives, surface active agents and the like in additionto the molecule of choice. Pharmaceutical compositions may also includeone or more active ingredients such as antimicrobial agents,antiinflammatory agents, anesthetics, and the like.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers,dispersing aids or binders may be desirable.

Some of the compositions may potentially be administered as apharmaceutically acceptable acid- or base-addition salt, formed byreaction with inorganic acids such as hydrochloric acid, hydrobromicacid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, andphosphoric acid, and organic acids such as formic acid, acetic acid,propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid,malonic acid, succinic acid, maleic acid, and fumaric acid, or byreaction with an inorganic base such as sodium hydroxide, ammoniumhydroxide, potassium hydroxide, and organic bases such as mono-, di-,trialkyl and aryl amines and substituted ethanolamines.

Methods of Treatment

Also disclosed are methods for treating a disease in a subject that iscaused or exacerbated by S100A9 activity, comprising administering tothe subject a composition comprising a CD33/S100A9 antagonist disclosedherein.

The innate immune system is crucial for initiation and amplification ofinflammatory responses. During this process, phagocytes are activated byPAMPs that are recognized by PRRs. Phagocytes are also activated byendogenous danger signals called alarmins or DAMPs via partly specific,partly common PRRs. Two members of the S100 protein family, S100A8 andS100A9, have been identified recently as important endogenous DAMPs. Thecomplex of S100A8 and S100A9 (also called calprotectin) is activelysecreted during the stress response of phagocytes. These molecules havebeen identified as endogenous activators of TLR4 and have been shown topromote lethal, endotoxin-induced shock. Importantly, S100A8/S100A9 isnot only involved in promoting the inflammatory response in infectionsbut was also identified as a potent amplifier of inflammation inautoimmunity as well as in cancer development and tumor spread. Thisproinflammatory action of S100A8/S100A9 involves autocrine and paracrinemechanisms in phagocytes, endothelium, and other cells. As a net result,extravasation of leukocytes into inflamed tissues and their subsequentactivation are increased. Thus, S100A8/S100A9 plays a pivotal roleduring amplification of inflammation.

Diseases that are associated with S100A8/S100A9 activity include, butare not limited to, rheumatoid arthritis, juvenile idiopathic arthritis,psoriatic arthritis, sepsis, atherosclerosis, acute coronarysyndrome/myocardial infarction, diabetes, psoriasis/inflammatory skindisease, inflammatory bowel disease, vasculitis, transplant rejection,SLE/glomerulonephritis, pancreatitis, cancer,dermatomyositis/polymyositis, and hyperzincemia/systemic inflammation.In addition, disease characterized by recurrent infections,hepato-splenomegaly, anemia, vasculopathie, concomitant cutaneousulcers, and systemic inflammation are defined by extraordinarily highlevels of S100A8/S100A9 in extracellular fluids.

In some embodiments, the method involves treating infection and/orpreventing sepsis in a patient in need thereof. Sepsis is caused by theimmune system's response to a serious infection, most commonly bacteria,but also fungi, viruses, and parasites in the blood, urinary tract,lungs, skin, or other tissues. There are number of microbial factorswhich can cause the typical septic inflammatory cascade. An invadingpathogen is recognised by its pathogen-associated molecular pattern(PAMP). Examples of PAMPs are lipopolysaccharides (LPS) in Gram-negativebacteria, flagellin in Gram-negative bacteria, muramyl dipeptide in thepeptidoglycan cell wall of a Gram-positive bacteria and CpG bacterialDNA. These PAMPs are recognized by the innate immune system's patternrecognition receptors (PRR). There are four families of PRRs: thetoll-like receptors, the C-type lectin receptors, the nucleotideoligemerization domain-like receptors and the RigI-helicases.S100A8/S100A9 acts as an endogenous TLR4 ligand involved inamplification of LPS effects on phagocytes upstream of TNF-α. AlthoughTNF-α is critical for LPS toxicity, blockade of TNF-α had a harmfulrather than protective effect in human sepsis. Moreover, in a smallstudy, S100A8/S100A9 levels were demonstrated to decrease in survivingpatients during recovery from sepsis, and nonsurvivors werecharacterized by high S100A8/S100A9 serum levels. Sepsis is usuallytreated with intravenous fluids and antibiotics. The disclosedCD33/S100A9 antagonist can be used instead of, or in addition to,intravenous antibiotics.

In some embodiments, the method involves treating an inflammatory and/orautoimmune disease in a patient in need thereof. S100A8/S100A9contributes to the pathogenesis of different types of arthritis.Macrophage-derived S100A8/S100A9 amplifies the inflammatory response inantigen-induced arthritis and also acts as endogenous DAMP in theabsence of infection or pathogenic agents. Autoimmunity is the failureof an organism in recognizing its own constituent parts as self, thusleading to an immune response against its own cells and tissues. Anydisease that results from such an aberrant immune response is termed anautoimmune or autoinflammatory disease. Autoimmune and autoinflammatorydiseases share common characteristics in that both groups of disordersresult from the immune system attacking the body's own tissues, and alsoresult in increased inflammation. Prominent examples include Celiacdisease, diabetes mellitus type 1 (IDDM), Sarcoidosis, systemic lupuserythematosus (SLE), Sjögren's syndrome, Churg-Strauss Syndrome,Hashimoto's thyroiditis, Graves' disease, idiopathic thrombocytopenicpurpura, Addison's Disease, rheumatoid arthritis (RA), gouty arthritis,Polymyositis (PM), Dermatomyositis (DM), graft-versus-host disease,pernicious anemia, Addison disease, scleroderma, Goodpasture's syndrome,inflammatory bowel diseases such as Crohn's disease, colitis, atypicalcolitis, chemical colitis; collagenous colitis, distal colitis,diversion colitis: fulminant colitis, indeterminate colitis, infectiouscolitis, ischemic colitis, lymphocytic colitis, microscopic colitis,gastroenteritis, Hirschsprung's disease, inflammatory digestivediseases, Morbus Crohn, non-chronic or chronic digestive diseases,non-chronic or chronic inflammatory digestive diseases; regionalenteritis and ulcerative colitis, autoimmune hemolytic anemia,sterility, myasthenia gravis, multiple sclerosis, Basedow's disease,thrombopenia purpura, insulin-dependent diabetes mellitus, allergy;asthma, atopic disease; arteriosclerosis; myocarditis; cardiomyopathy;glomerular nephritis; hypoplastic anemia; rejection after organtransplantation and numerous malignancies of lung, prostate, liver,ovary, colon, cervix, lymphatic and breast tissues, psoriasis, acnevulgaris, asthma, autoimmune diseases, celiac disease, chronicprostatits, glomerulonephritis, inflammatory bowel diseases, pelvicinflammatory disease, reperfusion injury sarcoidosis, vasculitis,interstitial cystitis, type 1 hypersensitivities, systemic sclerosis,dermatomyositis, polymyositis, and inclusion body myositis, andallergies. Treatments for autoimmune disease have traditionally beenimmunosuppressive, antiinflammatory (steroids), or palliative.Non-immunological therapies, such as hormone replacement in Hashimoto'sthyroiditis or Type 1 diabetes mellitus treat outcomes of theautoaggressive response, thus these are palliative treatments. Dietarymanipulation limits the severity of celiac disease. Steroidal or NSAIDtreatment limits inflammatory symptoms of many diseases. IVIG is usedfor CIDP and GBS. Specific immunomodulatory therapies, such as the TNFαantagonists (e.g. etanercept), the B cell depleting agent rituximab, theanti-IL-6 receptor tocilizumab and the costimulation blocker abatacepthave been shown to be useful in treating RA. Some of theseimmunotherapies may be associated with increased risk of adverseeffects, such as susceptibility to infection. The disclosed CD33/S100A9antagonist can be used instead of, or in addition to, these existingtreatments.

In some embodiments, the method involves treating a neurodegenerativedisease or disorder in a patient in need thereof. As used herein,“neurodegenerative disease” includes neurodegenerative diseaseassociated with protein aggregation, also referred to as “proteinaggregation disorders”, “protein conformation disorders”, or“proteinopathies”. Neurodegenerative disease associated with proteinaggregation include diseases or disorders characterized by the formationof detrimental intracellular protein aggregates (e.g., inclusions in thecytosol or nucleus) or extracellular protein aggregates (e.g., plaques).“Detrimental protein aggregation” is the undesirable and harmfulaccumulation, oligomerization, fibrillization or aggregation, of two ormore, hetero- or homomeric, proteins or peptides. A detrimental proteinaggregate may be deposited in bodies, inclusions or plaques, thecharacteristics of which are often indicative of disease and containdisease-specific proteins. For example, superoxide dismutase-1aggregates are associated with ALS, poly-Q aggregates are associatedwith Huntington's disease, and α-synuclein-containing Lewy bodies areassociated with Parkinson's disease.

Neurological diseases are also associated with immune failure related toincreasing levels of disease-causing factors that exceed the ability ofthe immune system to contain, or a situation in which immune functiondeteriorates or is suppressed concomitantly with disease progression,due to factors indirectly or directly related to the disease-causingentity. MDSCs can cause T-cell deficiency by suppressing effector T cellactivity, thus promoting neurodegenerative disease associated withimmune failure.

Representative examples of Protein Aggregation Disorders orProteopathies include Protein Conformational Disorders,Alpha-Synucleinopathies, Polyglutamine Diseases, Serpinopathies,Tauopathies or other related disorders. Other examples of neurologicaldiseases or include, but are not limited to, Amyotrophic LateralSclerosis (ALS), Huntington's Disease (HD), Parkinson's Disease (PD),Spinal Muscular Atrophy (SMA), Alzheimer's Disease (AD), diffuse Lewybody dementia (DLBD), multiple system atrophy (MSA), dystrophiamyotonica, dentatorubro-pallidoluysian atrophy (DRPLA), Friedreich'sataxia, fragile X syndrome, fragile XE mental retardation,Machado-Joseph Disease (MJD or SCA3), spinobulbar muscular atrophy (alsoknown as Kennedy's Disease), spinocerebellar ataxia type 1 (SCA1) gene,spinocerebellar ataxia type 2 (SCA2), spinocerebellar ataxia type 6(SCA6), spinocerebellar ataxia type 7 (SCA7), spinocerebellar ataxiatype 17 (SCA17), chronic liver diseases, familial encephalopathy withneuroserpin inclusion bodies (FENIB), Pick's disease, corticobasaldegeneration (CBD), progressive supranuclear palsy (PSP), amyotrophiclateral sclerosis/parkinsonism dementia complex, Cataract,serpinopathies, haemolytic anemia, cystic fibrosis, Wilson's Disease,neurofibromatosis type 2, demyelinating peripheral neuropathies,retinitis pigmentosa, Marfan syndrome, emphysema, idiopathic pulmonaryfibrosis, Argyophilic grain dementia, corticobasal degeneration, diffuseneurofibrillary tangles with calcification, frontotemporaldementia/parkinsonism linked to chromosome 17, Hallervorden-Spatzdisease, Nieman-Pick disease type C, subacute sclerosingpanencephalitis, cognitive disorders including dementia (associated withAlzheimer's disease, ischemia, trauma, vascular problems or stroke, HIVdisease, Parkinson's disease, Huntington's disease, Pick's disease,Creutzfeldt-Jacob disease, perinatal hypoxia, other general medicalconditions or substance abuse); delirium, amnestic disorders or agerelated cognitive decline; anxiety disorders including acute stressdisorder, agoraphobia, generalized anxiety disorder,obsessive-compulsive disorder, panic attack, panic disorder,post-traumatic stress disorder, separation anxiety disorder, socialphobia, specific phobia, substance-induced anxiety disorder and anxietydue to a general medical condition; schizophrenia or psychosis includingschizophrenia (paranoid, disorganized, catatonic or undifferentiated),schizophreniform disorder, schizoaffective disorder, delusionaldisorder, brief psychotic disorder, shared psychotic disorder, psychoticdisorder due to a general medical condition and substance-inducedpsychotic disorder; substance-related disorders and addictive behaviors(including substance-induced delirium, persisting dementia, persistingamnestic disorder, psychotic disorder or anxiety disorder; tolerance,dependence or withdrawal from substances including alcohol,amphetamines, cannabis, cocaine, hallucinogens, inhalants, nicotine,opioids, phencyclidine, sedatives, hypnotics or anxiolytics); movementdisorders, including akinesias and akinetic-rigid syndromes (includingParkinson's disease, drug-induced parkinsonism, postencephaliticparkinsonism, progressive supranuclear palsy, corticobasal degeneration,parkinsonism-ALS dementia complex and basal ganglia calcification),medication-induced parkinsonism (such as neuroleptic-inducedparkinsonism, neuroleptic malignant syndrome, neuroleptic-induced acutedystonia, neuroleptic-induced acute akathisia, neuroleptic-inducedtardive dyskinesia and medication-induced postural tremor), Gilles de laTourette's syndrome, epilepsy, and dyskinesias including tremor (such asrest tremor, postural tremor and intention tremor), chorea (such asSydenham's chorea, Huntington's disease, benign hereditary chorea,neuroacanthocytosis, symptomatic chorea, drug-induced chorea andhemiballism), myoclonus (including generalized myoclonus and focalmyoclonus), tics (including simple tics, complex tics and symptomatictics), and dystonia (including generalized dystonia such as iodiopathicdystonia, drug-induced dystonia, symptomatic dystonia and paroxysmaldystonia, and focal dystonia such as blepharospasm, oromandibulardystonia, spasmodic dysphonia, spasmodic torticollis, axial dystonia,dystonic writer's cramp and hemiplegic dystonia)]; obesity, bulimianervosa and compulsive eating disorders; pain including bone and jointpain (osteoarthritis), repetitive motion pain, dental pain, cancer pain,myofacial pain (muscular injury, fibromyalgia), perioperative pain(general surgery, gynecological), chronic pain, neuropathic pain,post-traumatic pain, trigeminal neuralgia, migraine and migraineheadache; obesity or eating disorders associated with excessive foodintake and complications associated therewith;attention-deficit/hyperactivity disorder; conduct disorder; mooddisorders including depressive disorders, bipolar disorders, mooddisorders due to a general medical condition, and substance-induced mooddisorders; muscular spasms and disorders associated with muscularspasticity or weakness including tremors; urinary incontinence;amyotrophic lateral sclerosis; neuronal damage including ocular damage,retinopathy or macular degeneration of the eye, hearing loss ortinnitus; emesis, brain edema and sleep disorders including narcolepsy,and apoptosis of motor neuron cells. Illustrative examples of theneuropathic pain include diabetic polyneuropathy, entrapment neuropathy,phantom pain, thalamic pain after stroke, post-herpetic neuralgia,atypical facial neuralgia pain after tooth extraction and the like,spinal cord injury, trigeminal neuralgia and cancer pain resistant tonarcotic analgesics such as morphine. The neuropathic pain includes thepain caused by either central or peripheral nerve damage. And itincludes the pain caused by either mononeuropathy or polyneuropathy.

In some cases, the method involves enhancing tumor immune response in apatient in need thereof. S100A8/S100A9 expression is increased inpatients with various tumors. Soluble factors secreted from tumor cellsare believed to induce overexpression of S100A8/S100A9, resulting inincreased generation of MDSCs. These MDSCs can inhibit anti-tumorresponses by CD8+ T cells and thus promote tumor growth. The cancer ofthe disclosed methods can be any cell in a subject undergoingunregulated growth, invasion, or metastasis. In some aspects, the cancercan be any neoplasm or tumor for which radiotherapy is currently used.In some aspects, the cancer can be any tumor that is resistant tostandard of care therapy. Thus, Also provided are methods of sensitizingtumors to standard care therapy, comprising administering to the subjectan effective amount of a compound or composition as disclosed herein.For example, the cancer can be a neoplasm or tumor that is notsufficiently sensitive to radiotherapy using standard methods. Thus, thecancer can be a sarcoma, lymphoma, leukemia, carcinoma, blastoma, orgerm cell tumor. A representative but non-limiting list of cancers thatthe disclosed compositions can be used to treat include lymphoma, B celllymphoma, T cell lymphoma, mycosis fungoides, Hodgkin's Disease, myeloidleukemia, bladder cancer, brain cancer, nervous system cancer, head andneck cancer, squamous cell carcinoma of head and neck, kidney cancer,lung cancers such as small cell lung cancer and non-small cell lungcancer, neuroblastoma/glioblastoma, ovarian cancer, pancreatic cancer,prostate cancer, skin cancer, liver cancer, melanoma, squamous cellcarcinomas of the mouth, throat, larynx, and lung, colon cancer,cervical cancer, cervical carcinoma, breast cancer, epithelial cancer,renal cancer, genitourinary cancer, pulmonary cancer, esophagealcarcinoma, head and neck carcinoma, large bowel cancer, hematopoieticcancers; testicular cancer; colon and rectal cancers, prostatic cancer,and pancreatic cancer. The disclosed CD33/S100A9 antagonist can be usedinstead of, or in addition to, existing antineoplastic drugs and/orradiation treatments.

As disclosed herein, LIN⁻HLA-DR⁻CD33⁺ MDSCs specifically accumulate inthe BM of myelodysplastic syndromes (MDS) patients and impairhematopoiesis through a mechanism that involves S100A9 as an endogenousligand for CD33 initiated signaling. Therefore, the disclosed method caninvolve treating MDS in a patient in need thereof. The myelodysplasticsyndromes (MDS) are hematological (blood-related) medical conditionswith ineffective production (or dysplasia) of the myeloid class of bloodcells. In some cases, the MDS patient has a chromosome 5q deletion(del(5q)). However, in other cases, the patient has non-del5q MDS.Although three agents are approved for the treatment of MDS in theUnited States (US), lenalidomide (LEN) represents the only targetedtherapeutic. Therefore, the disclosed CD33/S100A9 antagonist can be usedinstead of, or in addition to, lenalidomide.

In some embodiments, the method involves treating anemia of chronicdisease (including cancer-related anemia) in a patient, comprisingadministering to the subject an effective amount of a composition asdisclosed herein.

Administration

The disclosed CD33/S100A9 inhibitors may be administered in atherapeutically effective amount to a subject to treat a disease causedor exacerbated by S100A9 activity. The disclosed compositions, includingpharmaceutical composition, may be administered in a number of waysdepending on whether local or systemic treatment is desired, and on thearea to be treated. For example, the disclosed compositions can beadministered intravenously, intraperitoneally, intramuscularly,subcutaneously, intracavity, or transdermally. The compositions may beadministered orally, parenterally (e.g., intravenously), byintramuscular injection, by intraperitoneal injection, transdermally,extracorporeally, ophthalmically, vaginally, rectally, intranasally,topically or the like, including topical intranasal administration oradministration by inhalant.

Parenteral administration of the composition, if used, is generallycharacterized by injection. Injectables can be prepared in conventionalforms, either as liquid solutions or suspensions, solid forms suitablefor solution of suspension in liquid prior to injection, or asemulsions. A revised approach for parenteral administration involves useof a slow release or sustained release system such that a constantdosage is maintained. See, e.g., U.S. Pat. No. 3,610,795, which isincorporated by reference herein.

The compositions disclosed herein may be administered prophylacticallyto patients or subjects who are at risk for MDS. Thus, the method canfurther comprise identifying a subject at risk for MDS prior toadministration of the herein disclosed compositions.

The exact amount of the compositions required will vary from subject tosubject, depending on the species, age, weight and general condition ofthe subject, the severity of the allergic disorder being treated, theparticular nucleic acid or vector used, its mode of administration andthe like. Thus, it is not possible to specify an exact amount for everycomposition. However, an appropriate amount can be determined by one ofordinary skill in the art using only routine experimentation given theteachings herein. For example, effective dosages and schedules foradministering the compositions may be determined empirically, and makingsuch determinations is within the skill in the art. The dosage rangesfor the administration of the compositions are those large enough toproduce the desired effect in which the symptoms disorder are effected.The dosage should not be so large as to cause adverse side effects, suchas unwanted cross-reactions, anaphylactic reactions, and the like.Generally, the dosage will vary with the age, condition, sex and extentof the disease in the patient, route of administration, or whether otherdrugs are included in the regimen, and can be determined by one of skillin the art. The dosage can be adjusted by the individual physician inthe event of any counterindications. Dosage can vary, and can beadministered in one or more dose administrations daily, for one orseveral days. Guidance can be found in the literature for appropriatedosages for given classes of pharmaceutical products. For example,guidance in selecting appropriate doses for antibodies can be found inthe literature on therapeutic uses of antibodies, e.g., Handbook ofMonoclonal Antibodies, Ferrone et al., eds., Noges Publications, ParkRidge, N.J., (1985) ch. 22 and pp. 303-357; Smith et al., Antibodies inHuman Diagnosis and Therapy, Haber et al., eds., Raven Press, New York(1977) pp. 365-389. A typical daily dosage of the antibody used alonemight range from about 1 μg/kg to up to 100 mg/kg of body weight or moreper day, depending on the factors mentioned above.

Screening Methods

Also provided herein is a method of identifying an agent that can beused to treat MDS in a subject. The method can comprise providing asample comprising CD33 and S100A9 under conditions that allow CD33 andS100A9 to bind, contacting the sample with a candidate agent, detectingthe level of CD33/S100A9 binding, and comparing the binding level to acontrol, wherein a decrease in CD33/S100A9 binding compared to thecontrol identifies an agent that can be used to treat an inflammatorydisease.

The binding of S100A9 to CD33 can be detected using routine methods,such as immunodetection methods, that do not disturb protein binding.The methods can be cell-based or cell-free assays. The steps of varioususeful immunodetection methods have been described in the scientificliterature, such as, e.g., Maggio et al., Enzyme-Immunoassay, (1987) andNakamura, et al., Enzyme Immunoassays: Heterogeneous and HomogeneousSystems, Handbook of Experimental Immunology, Vol. 1: Immunochemistry,27.1-27.20 (1986), each of which is incorporated herein by reference inits entirety and specifically for its teaching regarding immunodetectionmethods. Immunoassays, in their most simple and direct sense, arebinding assays involving binding between antibodies and antigen. Manytypes and formats of immunoassays are known and all are suitable fordetecting the disclosed biomarkers. Examples of immunoassays are enzymelinked immunosorbent assays (ELISAs), radioimmunoassays (RIA),radioimmune precipitation assays (RIPA), immunobead capture assays,Western blotting, dot blotting, gel-shift assays, Flow cytometry,protein arrays, multiplexed bead arrays, magnetic capture, in vivoimaging, fluorescence resonance energy transfer (FRET), and fluorescencerecovery/localization after photobleaching (FRAP/FLAP).

In general, candidate agents can be identified from large libraries ofnatural products or synthetic (or semi-synthetic) extracts or chemicallibraries according to methods known in the art. Those skilled in thefield of drug discovery and development will understand that the precisesource of test extracts or compounds is not critical to the screeningprocedure(s) used.

Accordingly, virtually any number of chemical extracts or compounds canbe screened using the exemplary methods described herein. Examples ofsuch extracts or compounds include, but are not limited to, plant-,fungal-, prokaryotic- or animal-based extracts, fermentation broths, andsynthetic compounds, as well as modification of existing compounds.Numerous methods are also available for generating random or directedsynthesis (e.g., semi-synthesis or total synthesis) of any number ofchemical compounds, including, but not limited to, saccharide-, lipid-,peptide-, and nucleic acid-based compounds. Synthetic compound librariesare commercially available, e.g., from purveyors of chemical librariesincluding but not limited to ChemBridge Corporation (16981 Via Tazon,Suite G, San Diego, CA, 92127, USA, www.chembridge.com); ChemDiv (6605Nancy Ridge Drive, San Diego, CA 92121, USA); Life Chemicals (1103Orange Center Road, Orange, CT 06477); Maybridge (Trevillett, Tintagel,Cornwall PL34 0HW, UK)

Alternatively, libraries of natural compounds in the form of bacterial,fungal, plant, and animal extracts are commercially available from anumber of sources, including O2H, (Cambridge, UK), MerLionPharmaceuticals Pte Ltd (Singapore Science Park II, Singapore 117528)and Galapagos NV (Generaal De Wittelaan L11 A3, B-2800 Mechelen,Belgium).

In addition, natural and synthetically produced libraries are produced,if desired, according to methods known in the art, e.g., by standardextraction and fractionation methods or by standard synthetic methods incombination with solid phase organic synthesis, micro-wave synthesis andother rapid throughput methods known in the art to be amenable to makinglarge numbers of compounds for screening purposes. Furthermore, ifdesired, any library or compound, including sample format anddissolution is readily modified and adjusted using standard chemical,physical, or biochemical methods. In addition, those skilled in the artof drug discovery and development readily understand that methods fordereplication (e.g., taxonomic dereplication, biological dereplication,and chemical dereplication, or any combination thereof) or theelimination of replicates or repeats of materials already known fortheir effect on MDS should be employed whenever possible.

Candidate agents encompass numerous chemical classes, but are most oftenorganic molecules, e.g., small organic compounds having a molecularweight of more than 100 and less than about 2,500 Daltons. Candidateagents can include functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and typicallyinclude at least an amine, carbonyl, hydroxyl or carboxyl group, forexample, at least two of the functional chemical groups. The candidateagents often contain cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups.

In some embodiments, the candidate agents are proteins. In some aspects,the candidate agents are naturally occurring proteins or fragments ofnaturally occurring proteins. Thus, for example, cellular extractscontaining proteins, or random or directed digests of proteinaceouscellular extracts, can be used. In this way libraries of procaryotic andeucaryotic proteins can be made for screening using the methods herein.The libraries can be bacterial, fungal, viral, and vertebrate proteins,and human proteins.

Definitions

The term “antibody” refers to natural or synthetic antibodies thatselectively bind a target antigen. The term includes polyclonal andmonoclonal antibodies. In addition to intact immunoglobulin molecules,also included in the term “antibodies” are fragments or polymers ofthose immunoglobulin molecules, and human or humanized versions ofimmunoglobulin molecules that selectively bind the target antigen.

A “chimeric molecule” is a single molecule created by joining two ormore molecules that exist separately in their native state. The single,chimeric molecule has the desired functionality of all of itsconstituent molecules.

A “fusion protein” refers to a polypeptide formed by the joining of twoor more polypeptides through a peptide bond formed between the aminoterminus of one polypeptide and the carboxyl terminus of anotherpolypeptide. The fusion protein can be formed by the chemical couplingof the constituent polypeptides or it can be expressed as a singlepolypeptide from nucleic acid sequence encoding the single contiguousfusion protein. A single chain fusion protein is a fusion protein havinga single contiguous polypeptide backbone. Fusion proteins can beprepared using conventional techniques in molecular biology to join thetwo genes in frame into a single nucleic acid, and then expressing thenucleic acid in an appropriate host cell under conditions in which thefusion protein is produced.

The term “inhibit” refers to a decrease in an activity, response,condition, disease, or other biological parameter. This can include butis not limited to the complete ablation of the activity, response,condition, or disease. This may also include, for example, a 10%reduction in the activity, response, condition, or disease as comparedto the native or control level. Thus, the reduction can be a 10, 20, 30,40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between ascompared to native or control levels.

A “liposome” is a small vesicle composed of various types of lipids,phospholipids and/or surfactant which is useful for delivery of a drug(such as the antagonists disclosed herein and, optionally, achemotherapeutic agent) to a mammal. The components of the liposome arecommonly arranged in a bilayer formation, similar to the lipidarrangement of biological membranes.

The terms “peptide,” “protein,” and “polypeptide” are usedinterchangeably to refer to a natural or synthetic molecule comprisingtwo or more amino acids linked by the carboxyl group of one amino acidto the alpha amino group of another.

The term “percent (%) sequence identity” or “homology” is defined as thepercentage of nucleotides or amino acids in a candidate sequence thatare identical with the nucleotides or amino acids in a reference nucleicacid sequence, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity. Alignmentfor purposes of determining percent sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN,ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters formeasuring alignment, including any algorithms needed to achieve maximalalignment over the full-length of the sequences being compared can bedetermined by known methods.

The term “pharmaceutically acceptable” refers to those compounds,materials, compositions, and/or dosage forms which are, within the scopeof sound medical judgment, suitable for use in contact with the tissuesof human beings and animals without excessive toxicity, irritation,allergic response, or other problems or complications commensurate witha reasonable benefit/risk ratio.

The term “specifically binds”, as used herein, when referring to apolypeptide (including antibodies) or receptor, refers to a bindingreaction which is determinative of the presence of the protein orpolypeptide or receptor in a heterogeneous population of proteins andother biologics. Thus, under designated conditions (e.g. immunoassayconditions in the case of an antibody), a specified ligand or antibody“specifically binds” to its particular “target” (e.g. an antibodyspecifically binds to an endothelial antigen) when it does not bind in asignificant amount to other proteins present in the sample or to otherproteins to which the ligand or antibody may come in contact in anorganism. Generally, a first molecule that “specifically binds” a secondmolecule has an affinity constant (Ka) greater than about 10⁵ M⁻¹ (e.g.,10⁶ M⁻¹, 10⁷ M⁻¹, 108 M⁻¹, 10⁹ M⁻¹, 10¹⁰ M⁻¹, 10¹¹ M⁻¹, and 10¹² M⁻¹ ormore) with that second molecule.

The term “subject” refers to any individual who is the target ofadministration or treatment. The subject can be a vertebrate, forexample, a mammal. Thus, the subject can be a human or veterinarypatient. The term “patient” refers to a subject under the treatment of aclinician, e.g., physician.

The term “therapeutically effective” refers to the amount of thecomposition used is of sufficient quantity to ameliorate one or morecauses or symptoms of a disease or disorder. Such amelioration onlyrequires a reduction or alteration, not necessarily elimination.

The term “treatment” refers to the medical management of a patient withthe intent to cure, ameliorate, stabilize, or prevent a disease,pathological condition, or disorder. This term includes activetreatment, that is, treatment directed specifically toward theimprovement of a disease, pathological condition, or disorder, and alsoincludes causal treatment, that is, treatment directed toward removal ofthe cause of the associated disease, pathological condition, ordisorder. In addition, this term includes palliative treatment, that is,treatment designed for the relief of symptoms rather than the curing ofthe disease, pathological condition, or disorder; preventativetreatment, that is, treatment directed to minimizing or partially orcompletely inhibiting the development of the associated disease,pathological condition, or disorder; and supportive treatment, that is,treatment employed to supplement another specific therapy directedtoward the improvement of the associated disease, pathologicalcondition, or disorder.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

EXAMPLES Example 1: Microenvironment Induced Myelodysplasia Mediated byMyeloid-Derived Suppressor Cells

Methods

MDS patients. The majority of patients with MDS were low risk unlessotherwise specified. All patients were confirmed by central review andclassified in accordance with either the World Health Organizationcriteria or International Prognostic Scoring System (IPSS). Patientswere recruited from the Malignant Hematology clinic at the H. LeeMoffitt Cancer Center & Research Institute and the Radboud UniversityNijmegen Medical Centre, Department of Hematology in the Netherlands.Bone marrow mononuclear cells (BM-MNC) were isolated from heparinized BMaspirates by Ficoll-Hypaque gradient centrifugation, as previouslydescribed (Wei, S., et al. 2009. Proc Natl Acad Sci USA106:12974-12979). MDSCs were defined and purified by fluorescenceactivated cell sorting (FACS) of CD33⁺ cells lacking expression oflineage (Lin⁻) markers (CD3, CD14, CD16, CD19, CD20, CD56) and HLA-DR.

Mice. All mouse work was approved by the Institutional Animal Care andUse Committee at the University of South Florida. Wild type (WT) FVB/NJmice were purchased from Jackson Laboratories, and S100A9 knockout mice(KO) and S100A9 transgenic mice (Tg) were generated and used aspreviously described (Cheng, P., et al. 2008. J Exp Med 205:2235-2249;Manitz, M. P., et al. 2003. Mol Cell Biol 23:1034-1043). S100A9Tg micewere generated from FVB/NJ homozygous mice and bred for more than 15generations. For the competitive transplant experiment, 18 week oldFVB/NJ wild type female mice were irradiated once in a rotating gammairradiator for a total dose of 900Gy. Concurrently, BM cells wereisolated from the tibias and femurs of age-matched male WT or S100A9Tgmice from which HSCs were then enriched by magnetic cell sorting (MACS,Millitenyi Biotech) following the manufacturer's protocol. Six hourspost-irradiation 1×10⁷ enriched HSCs were given by tail vein injectioninto recipient mice. Mice were then monitored every other day withweight measurements under a sterile hood. Peripheral blood for CBC wascollected from the antero-orbital vein by Vivarium staff at the centerweekly. By 8 weeks (after which the WT recipients were engrafted, WBC>3×10³ cells/ml of blood) mice were euthanized by CO₂ aspiration atwhich point peripheral blood was collected by heart puncture followed bydissection of tibias and femurs (as before) and spleen for assessment.

Fluorescence in situ hybridization (FISH). FISH was done at theCytogenetics Laboratory of Moffitt Cancer Center, and detailed methodshave been previously described (Wei, S., et al. 2009. Proc Natl Acad SciUSA 106:12974-12979). Target DNA from MDSC positive (MDS-MDSC) cells andMDSC negative cells was purified from the same patients who werepreviously confirmed to have del5q or del7q using a commerciallyavailable test (Abbott laboratory).

Immunostaining. BM-MNCs were purified from MDS patients, diluted to aconcentration of 3×10⁵ cells/ml, cytospinned onto microscope slides andfixed with methanol/acetone (3:1 ratio at −20° C. for 30 min). Washeswere done with triton X-100 buffer for 5 min and 50 mM Tris Buffer (pH7.4) for 10 min prior to blocking for non-specific binding with serum.She slides were stained with the primary antibodies: rabbit anti-CD33antibody (1:100 dilution, Santa Cruz), mouse anti-granzyme B (1:100dilution, Fitzgerald Industries) and mouse anti-human CD71 (1:100dilution, BD Biosciences) followed by their respective secondaryantibodies, AlexaFluor-594 goat anti-rabbit IgG (Invitrogen), FITC goatanti-mouse (Sigma) and Alexa-350 goat anti-mouse IgG (Molecular Probes).Rat anti-human glycophorin A was pre-conjugated to Alexa-647 using a kitfrom Molecular Probes) before addition to the sample (1:50 dilution, AbDSerotec) followed by mounting the slides with aqueous medium (MolecularProbes, USA). Immunofluorescence was detected using a Zeiss automatedupright fluorescence microscope and images captured by a Nikon camerawith the Capture Program AxioVision. Detailed methods forimmuno-staining on S100A9 transfected SJCRH30 cells have been publishedpreviously (Chen, X., et al. 2008. Blood 113(14):3226-34). Specifically,SJCRH30 cells, which lack detectable expression levels of both CD33 andS100A9, were transfected with either S100A9 or S100A8 (negative control)for 48-72 hours. After incubation with CD33-fusion for 30 min (2 μg/ml),1×10⁴ cells were cytospinned onto slides and then stained with asecondary anti-human IgG1-APC before analysis by immunofluorescencemicroscopy. Similarly, SJCRH30 cells, stable-transfected with CD33, wereincubated with rhS100A9 tagged with DDK for various time points andstained by the same methodology before analysis.

Suppression assays. To determine whether MDSCs are capable of mediatingT cell suppression, CD45⁺ CD33⁺ CD11b⁺ Lin cells were sorted from fullbone marrow of MDS patients by FACS. The following antibodies were used:CD45-PECy7, CD33-PECy5, CD11b-FITC, CD3-PE, CD14-PE, CD20-PE (allBeckman Coulter, Fullerton, CA, USA), CD16-PE, CD19-PE (DAKO, Glostrup,Denmark) and CD56-PE (BD Biosciences, San Diego, CA, USA). T cells wereisolated by Magnetic Activated Cell Sorting (MACS) using CD3 microbeads(Miltenyi Biotec, Aubern, CA, USA) from autologous peripheral blood.20,000 T cells were seeded in a 96 wells round bottom plate intriplicate in Iscove's modified Dulbecco medium (IMDM; Invitrogen,Carlsbad, CA) supplemented with 10% human serum PAA (PAA Laboratories,Pasching, Austria). Cultures were stimulated with 30 U/ml IL-2 (Chiron,Emeryville, CA, USA), and anti-CD3/anti-CD28 coated beads (Invitrogen,Carlsbad, CA, USA) at a 1:2 ratio of T cells to beads. MDSCs wereadmixed with T cell cultures at ratios of 1:2 and 1:4 and supplementedwith 10 ng/ml GM-CSF to support MDSC viability. After 3 days ofco-culture, culture supernatants were harvested to measure IFN-γconcentration by ELISA (Pierce Endogen, Rockford, IL, USA).Subsequently, 0.5 μCi ³H-thymidine (Perkin Elmer, Groningen, theNetherlands) was added to each well and, after overnight incubation,³H-thymidine incorporation was measured using a 1205 Wallac Betaplatecounter (PerkinElmer). To determine whether differences in proliferationand IFN-γ production were statistically significant, one-way Anova withBonferroni post-hoc test was used. Statistical significance was acceptedfor p values <0.05.

Colony-forming Assay. Cells isolated from either human BM, or fromS100A9Tg, S100A9KO or WT tibias and femurs were subjected to ACK for 5min at room temperature (Sigma) to lyse the red blood cells. RemainingBM cells were then seeded into complete methylcellulose media (MethoCultcomplete medium with necessary cytokines and growth factors (StemCellTechnologies) and the mixture was placed in duplicate gridded 35-mmculture dishes (2×10⁵ cells/dish) and incubated at 37° C. in 5% CO₂ for7-14 days. After incubation, colonies of BFU-E and CFU-GM wereidentified manually and counted using an inverted light microscope. Forthe colony formation assays performed using ATRA treated mice, weadministered ATRA at 250 μg (200 μl) or vehicle (Olive oil) orally forfive consecutive days before resting two days.

mRNA expression by Real-time Quantitative. RT-PCR and quantitativeRT-PCR (qRT-PCR) reactions were performed by means of iQ SYBR GreenSupermix (Bio-Rad). The reaction mixture (25 μl total) contained 12.5 μliQ SYBR green supermix, 0.25 μl forward primer (s GAPDH) (20 μM), 1 μlRNase-free water, and 1.0 μl cDNA. The following cycles were performed1×3 min at 95° C., 40 amplification cycles (15 s 95° C., 60 s 56° C.),1×1 min 95° C., 1×1 negative control without cDNA template was run withevery assay. The optimal melting point of dsDNA I and min 55° C. and amelting curve (80×10 s 55° C. with an increase of 0.5° C. per 10seconds). The efficiency of the reaction was optimized beforehand.Transcript copy number per individual was calculated by normalization toGAPDH expression. The relative level of gene expression for each patientwas calculated by normalization to the average expression level observedin five controls. CD33 transfected and un-transfected SJCRH 30 cellswere treated with 1 μg of rhS100A9 for 20 min and the expressionmeasured by Q-PCR for the presence of IL10 and TGFβ from total RNA andcalculated by the ΔΔCt method where rhS100A9 untreated cells were theexperimental control and the housekeeping gene GAPDH was the internalcontrol. Error bars represent the SEM of three separate experiments.

Preparation of the CD33/Siglec 3 chimeric fusion protein. Recombinantsoluble fusion of CD33/Siglec 3 ectodomain were constructed as describedpreviously (Cannon, J. P., et al. 2012. Immunogenetics 64:39-47; Cannon,J. P., et al. 2011. Methods Mol Biol 748:51-67; Cannon, J. P., et al.2008. Immunity 29:228-237). Specifically, cDNA fragments encodingCD33/Siglec 3 ectodomain were amplified by PCR and inserted into avector that encodes the human Fcγ followed by a c-terminal recognitionsite for E. coli biotin ligase. This vector has been engineered tofacilitate the fusion of gene segments encoding extracellular Ig-typedomains to the Fc region of human IgG1. The recombinant proteins wereexpressed in 293T cells post-transfection, using Lipofectamine(Invitrogen), with three successive harvests of 25 ml OPTI-MEM Iserum-free medium. The harvests were pooled, centrifuged at 500 g for 10min to remove debris and stored at 4° C. in 0.02% sodium azide.Concentrations of CD33-fusions in culture supernatants were determinedby Bradford assay (Biorad, Carlsbad, CA).

Mass spectrometry. Following in-gel tryptic digestion, peptides wereextracted and concentrated under vacuum centrifugation. A nanoflowliquid chromatograph (Easy-nLC, Proxeon, Odense, Denmark) coupled to anelectrospray ion trap mass spectrometer (LTQ, Thermo, San Jose, CA) wasused for tandem mass spectrometry peptide sequencing experiments. Thesample was first loaded onto a trap column (BioSphere C18 reversed-phaseresin, 5 μm, 120 Å, 100 μm ID, NanoSeparations, Nieuwkoop, Netherlands)and washed for 3 minutes at 8 ml/minute. The trapped peptides wereeluted onto the analytical column, (BioSphere C18 reversed-phase resin,150 mm, 5 μm, 120 Å, 100 μm ID, NanoSeparations, Nieuwkoop,Netherlands). Peptides were eluted in a 60 minute gradient from 5% B to45% B (solvent A: 2% acetonitrile+0.1% formic acid; solvent B: 90%acetonitrile+0.1% formic acid) with a flow rate of 300 nl/min. Fivetandem mass spectra were collected in a data-dependent manner followingeach survey scan. Sequences were assigned using Mascot(www.matrixscience.com) searches against human IPI

entries. Carbamidomethylation of cysteine, methionine oxidation, anddeamidation of asparagine and glutamine were selected as variablemodifications, and as many as 2 missed tryptic cleavages were allowed.Precursor mass tolerance is set to 2.5 and fragment ion tolerance to0.8. Results from Mascot were compiled in Scaffold, which was used formanual inspection of peptide assignments and protein identifications.

Identifications of specific binding of S100A9 to CD33/Siglec 3. ELISAassay for CD33-fusion binding cell lysate of S100A9 transfected SJCRH 30cells. Ninety-six well flat-bottom ELISA plates were coated overnightwith lug/ml of monoclonal anti-S100A9, as per the manufacturer'ssuggestions. After washing with 1 X PBS-T, 50 μl of lysates fromun-transfected cells (negative) or S100A9 transfected cells was added tothe wells. Secondary antibody was either a S100A9 polyclonal antibody(positive control) or CD33-fusion as indicated followed by ELISA HRPreaction analysis at 440 nm.

Preparation of adenoviral vector expressing CD33. CD33 plasmid(GeneCopeia) was subcloned into a pShuttle-IRES-hrGFP-1 vector(containing the CMV promoter and hrGFP). The PmeI-digested shuttlevectors were then co-transformed into electro-competent BJ5183 bacteriawith pAdEasy-1 (containing the viral backbone) and selected on KanamycinLB plates. The plasmid in the bacteria was amplified and purified usinga plasmid maxiprep system (Qiagen). The complete adenoviral vector waslinearized by PacI digestion and then transfected into AD293 cells usingLipofectamine (Invitrogen). All recombinant adenoviruses were amplifiedin AD293 cells. Viral stocks were obtained by amplification of the AD293cells followed by standard two-step CsCl gradient ultracentrifugation,dialysis, and storage in a glycerol stock (10% volume/volume) at −80° C.The titer of each viral stock was routinely tested to be 10⁻¹-10¹² pfuby plaque forming assay using AD293 cells.

Preparation of shRNA lentiviral vectors. SureSilencing™ shRNA Plasmidfor Human S100A8, S100A9 and CD33 and negative control (non-target) werepurchased from SABiosciences. 293T cells were transfected usingtransfection reagent (SABiosciences,) according to manufacturer'sinstructions. Following 6 hours of incubation, the transfection reagentwas removed and replaced with fresh DMEM supplemented with 10% fetalbovine serum. Virus containing medium was collected 24-48 hr later.Plasmids, pcDNA3 wild-type DAP12 and P23-DAP12 were cut with HindIII andXhoI (Promega), and DNA Polymerase I Large (KlenowI) (New EnglandBiolabs Inc.) was used to fill in recessed 3′ ends of DNA fragments.pWPI, which contains a GFP expression cassette (Addgene Organization),was digested using PMEI (New England Biolabs Inc.). After cutting DNAand vector, pWPI were purified by Strata Prep PCR purification kit(Qiagen), DNAs were ligated (Takara DNA ligation kit, Fisher) intovector pWPI and STBL2 competent cells were transformed (Invitrogen).293T cells were transfected with pWPI lentivirus vector, the packagingplasmid, psPAX2, and the envelope plasmid, pMD2.G (Addgene Organization)using the Lipofectemine-2000 (Invitrogen) at a ratio of 4:3:1 accordingto standard protocols. Following 6 hours of incubation, the transfectionreagent was removed and replaced with fresh DMEM supplemented with 10%fetal bovine serum. Virus containing medium was collected 24-48 hrlater. Cell infection was performed as described above. Four days afterthe first infection, transduced cells were isolated by FACS sorting GFP⁺cells with >99% purity.

Infection of MDSC from MDS patients. MDSCs isolated from MDS patientswere infected three times using virus-containing infection medium at 24hr intervals in the presence of 8 μg/ml polyberene. For each infection,cells were plated in 12-well dishes at 1×10⁶ cells/well. Four days afterthe first infection, cells were harvested and used for real time PCR,Western blot analysis, flow cytometry, or colony formation assays.

Flow cytometry. BM-MNCs were stained with appropriate specificconjugated antibodies in PBS with 2% BSA buffer. For MDSC sorting, FITCanti-CD3 and FITC anti-HLA-DR were used as positive controls and isotypeIgG were used for negative control and to detect non-specific staining.Cells were gently mixed and incubated for 30 min at 4° C. in the dark.Samples were washed with PBS and centrifuged at 500 g for 5 min. Forcells used in phenotypic analysis only, 0.5 ml of 1% paraformaldehyde inPBS was added prior to analysis.

Cells were washed in PBS and then stained with PE-conjugated mAbsspecific to CD40, CD80, CD83, CD86, CCR7, CD11c, CD14, HLA-DR, TLR2, orTLR4 and relevant isotype controls (eBioscience) for 30 min in the dark,on ice. The cells were then washed with PBS containing 0.5% BSA. Livecells were gated based on negative staining for 7-AAD. Samples wereacquired on a FACSCalibur flow cytometer and the analysis was performedusing Flowjo 6.3.4 software.

Arginase activity. Arginase activity was measured in cell lysates aspreviously described (Youn, J. I., et al. 2008. J Immunol181:5791-5802). In brief, cells were lysed for 30 min with 100 μl of0.1% Triton X-100. Subsequently, 100 μl of 25 mM Tris-HCl and 10 μl of10 mM MnCl2 were added, and the enzyme was activated by heating for 10min at 56° C. Arginine hydrolysis was conducted by incubating the lysatewith 100 μl of 0.5 M L-arginine (pH 9.7) at 37° C. for 120 min. Thereaction was stopped with 900 μl of H₂SO₄ (96%)/H₃PO₄ (85%)/H₂O (1/3/7,v/v/v). Urea concentration was measured at 540 nm after addition of 40μl of α-isonitrosopropiophenone (dissolved in 100% ethanol), followed byheating at 95° C. for 30 min.

NO production. Equal volumes of culture supernatants (100 μl) were mixedwith Greiss reagent solution (1% sulfanilamide in 5% phosphoric acid and0.1% N-1-naphthylethylenediamine dihydrochloride) in double-distilledwater and incubated for 10 min at room temperature. Absorbance of themixture was measured at 550 nm using a microplate plate reader(Bio-Rad). Nitrite concentrations were determined by comparing theabsorbance values for the test samples to a standard curve generated byserial dilution of 0.25 mM sodium nitrite.

Western Blot analysis. Cell lysates were prepared by resuspending cellpellet in 1% NP-40, 10 mM Tris, 140 mM NaCl, 0.1 mM PMSF, 10 mMiodoacetamide, 50 mM NaF, 1 mM EDTA, 0.4 mM sodium orthovanadate, 10μg/ml leupeptin, 10 μg/ml pepsatin, and 10 μg/ml aprotinin and lysing onice for 30 min. Cell lysates were centrifuged at 12,000 g for 15 min toremove nuclei and cell debris. The protein concentration of the solubleextracts was determined by using the Bio-Rad (Bradford) protein assay.50 μg of protein (per lane) was separated on a 10% SDS-polyacrylamidegel by electrophoresis then transferred to a PVDF membrane. Membraneswere probed for indicated antibody: anti-S100A8 or anti-S100A9 (MRP8 orcalgranulin A and anti-MRP14 or calgranulin B respectively, Santa Cruz);anti-S100A8/A9 (Santa Cruz); anti-phospho Erk, anti-total Erk,anti-phospho Syk, and anti-total Syk (Cell Signaling). Proteins weredetected with the enhanced chemiluminescence detection system (ECL,Amersham). Lysate from S100A9 un-transfected, empty vector and S100A9transfect SJCRH30 or AD293 cells was serially-diluted (as indicated inthe figure) with the first lysate, starting at 50 μg, followed byloading onto a nitrocellulose membrane and blocked overnight at 4° C. in5% milk. Following incubation with CD33-fusion for 2 hours at roomtemperature, and staining with an anti-human IgG HRP-conjugatedsecondary, the membrane was washed and used for radiograph analysis todemonstrated specific S100A9 binding to CD33. The membrane wasafterwards used for Coomassie blue staining to show the relative equalloading of proteins on each dot in the nitrocellulose membrane.

Complete peripheral blood cell counts (CBC). CBC was performed by animalcore laboratory pathological personnel in the vivarium at the MoffittCancer Center. The mouse blood parameters were determined as describedin Table 1 with a Heska Hematrue Hematology Analyzer.

Pathological examination of spleen and BM biopsy from wt and S100A9Tgmice. Bone marrow cells were obtained from bilateral tibiae and femurafrom 6 month old S100A9Tg and wt control mice as previously described(Xu, S., et al. 2010. J Biomed Biotechnol 2010:105940). Touch prints ofmurine splenocytes were prepared as described (Joachim, H. L., et al.2008. Iochim's lymph node pathology. Chapter 3. cytopathology. WoltersKluwer/Lippincott Williams & Wilkins.:p 21-22). Bone marrow aspiratesand touch-imprints were stained using Wright-Giemsa stain. Sections ofbone marrow and spleen were fixed in 10% phosphate-buffered neutralformalin, decalcified (only applied to bone marrow) and embedded inparaffin by routine procedures. Sections were cut at 4 μm and stainedwith hematoxylin and eosin (H&E) and periodic acid Schiff (PAS). Thepresence of myelodysplastic features characteristic of MDS was evaluatedby experienced hematopathologist. BM core biopsy shows 50% cellularitywith maturing trilineage hematopoiesis (H&E, 200×). FIG. 5E shows a highpower view of the BM biopsy that demonstrates normal appearingmegakaryocytes with normal lobation. Mixed myeloid and erythroidprecursors are normally distributed with estimated M:E ratio of 2:1(H&E, 600×). Wright-Giemsa stained BM aspirate exhibits full maturationin all three lineages without dysplastic features (Wright-Giemsa,1000×). Inlet shows a normal lobated megakaryocyte (FIG. 5F). Touchimprint of mouse spleen displays predominance of small and matureappearing lymphocytes intermingled with occasional erythropoieticprecursors (Wright-Giemsa, 1000×) (FIG. 5G). BM core biopsy revealshypercellularity, approximately 95% with increased megakaryocytes,especially in small forms (H&E, 200×) (FIG. 5H). High powermagnification highlights dysplastic megakaryocytes with single orhypolobated, or disjointed nuclei and markedly increased in number (H&E,600×) (insert) (FIG. 5I). Inlet includes two markedly dysplasticmicromegakaryocytes with hypolobation. BM aspirates exhibits mildlyincreased blasts admixed with myeloid and erythroid precursors. Thelatter demonstrates slightly irregular nuclear contour and minimalmegaloblastoid changes (Wright-Giemsa, 1000×) (FIG. 5J). Inlet containstwo blasts showing delicate or fine chromatin, prominent nucleoli, highN:C ratio, and scant basophilic cytoplasm. Touch preparation oftransgenic mouse spleen show increased erythroid precursors; some ofthem displaying enlarged size with abnormal nuclearity and occasionalnuclear bridge (Wright-Giemsa, 1000×) (FIG. 5K).

Statistics. All data was presented as means±SEM. Statisticalcalculations were performed with Microsoft Excel or GraphPad Prismanalysis tools. Differences between individual groups were analyzed bypaired t-test. P values of <0.05 were considered to be statisticallysignificant.

Results

Lin⁻HLA-DR⁻CD33⁺ MDSC are expanded in MDS primary BM specimens anddirect suppression of autologous erythroidprecursors. Bone marrowmononuclear cells (BM-MNC) were isolated from MDS BM aspirates (n=12),age-matched healthy BM (n=8), or non-MDS cancer patients (4 breast and 4lymphoma) and analyzed for the presence of LIN⁻HLA-DR⁻CD33⁺ MDSCs byflow cytometry. MDS patients exhibited markedly higher numbers of MDSCs(median 35.5%, P<0.0001) compared to healthy donors or non-MDS cancerpatients (less than 5%, FIG. 1A). To determine if MDS-MDSCs are derivedfrom the malignant MDS clone, LIN⁻HLA-DR⁻CD33⁺ MDSCs were sorted fromMDS specimens with chromosome 5q [del(5q)] or 7q [del(7q)] deletion andanalyzed by fluorescence in situ hybridization (FISH) with specificprobes.

Cytogenetically abnormal cells harboring del(5q) or del(7q) wererestricted to the non-MDSC population, whereas LIN⁻HLA-DR⁻CD33⁺ MDSCsdisplayed a corresponding normal chromosome complement (FIG. 1B). Exomesequencing studies have shown that clonal somatic gene mutations aredemonstrable in the vast majority of MDS specimens lacking chromosomeabnormalities by metaphase karyotyping. To further evaluate therelationship between MDSC and the MDS clone, a QPCR array of the mostcommon gene mutations in MDS (Qiagen) was performed in purified MDSC andnon-MDSC populations from primary bone marrow MDS specimens. Mutationsinvolving CBL, EZH2, IDH1/2, N-RAS, SRSF2, U2A535 and RUNX1 genes weredetected in the MDS specimens, however, all mutations were restricted tothe MDSC-depleted fraction (Table 1), indicating that LIN⁻HLA-DR⁻CD33⁺MDSC are distinct from the malignant clone.

TABLE 1 qBiomarker ™ Somatic Mutation PCR Array Human MyelodysplasticSyndromes (n = 6) NON-MDSC COSMIC Pt Pt Pt Pt Pt P MDSC Gene ID ntchange AA change 1 2 3 4 5 6 N = 6 ASXL1 36166 c.1772_1773insAp.Y591fs*1 − − − − − − − ASXL1 41716 c.1888_1909del22 p.H630fs*66 − − −− − − − ASXL1 41717 c.2302C > T p.Q768* − − − − − − − ASXL1 52930c.2324T > G p.L775* − − − − − − − ASXL1 41715 c.3202C > T p.R1068* − − −− − − − CBL 34052 c.1111T > C p.Y371H − − − − − − − CBL 34055 c.1139T >C p.L380P − + + + + + − CBL 34057 c.1150T > C p.C384R − + + + − + − CBL34077 c.1259G > A p.R420Q − − − − + − − DNMT3A 53042 c.2644C > T p.R882C− − − − − − − DNMT3A 87007 c.2711C > T p.P904L − − − − − − − EZH2 37031c.1936T > A p.Y646N − + + + − + − EZH2 37029 c.1937A > C p.Y646S − + + +− + − EZH2 37028 c.1937A > T p.Y646F − + + + + + − IDH1 28748 c.394C > Ap.R132S − − − − − − − IDH1 28749 c.394C > G p.R132G − + + + + + − IDH128747 c.394C > T p.R132C − + − − + + − IDH1 28746 c.395G > A p.R132H− + + + − − − IDH1 28750 c.395G > T p.R132L − + + + + + − IDH2 41877c.418C > T p.R140W − + + + − − − IDH2 41590 c.419G > A p.R140Q + − −− + + − IDH2 41875 c.419G > T p.R140L − + + + + + − IDH2 34039 c.514A >T p.R172W + + + + + + − IDH2 33733 c.515G > A p.R172K − + + + + + − IDH233732 c.515G > T p.R172M − + + + − + − IDH2 34090 c.516G > T p.R172S −− + − − − − NRAS 580 c.181C > A p.Q61K − + + + + + − NRAS 584 c.182A > Gp.Q61R − + + + − + − NRAS 583 c.182A > T p.Q61L − − − − − − − NRAS 586c.183A > C p.Q61H − + + + + + − NRAS 585 c.183A > T p.Q61H − − − − + −NRAS 563 c.34G > A p.G12S − + − − − + − NRAS 562 c.34G > T p.G12C− + + + + − − NRAS 564 c.35G > A p.G12D − − + − + + − NRAS 565 c.35G > Cp.G12A − + − + + + − NRAS 566 c.35G > T p.G12V − + + + + + − NRAS 569c.37G > C p.G13R − + + + + + − NRAS 570 c.37G > T p.G13C − + + + + + −NRAS 573 c.38G > A p.G13D − − − − − − − NRAS 574 c.38G > T p.G13V − +− + + + − RUNX1 24756 c.167T > C p.L56S − + + − − + − RUNX1 24736c.319C > T p.R107C − + + + − + − RUNX1 24769 c.496C > T p.R166* − − − −− − − RUNX1 24721 c.592G > A p.D198N − − − − − − − RUNX1 24799 c.593A >G p.D198G − − − − + − − RUNX1 24805 c.602G > A p.R201Q − + − + − − −RUNX1 24731 c.611G > A p.R204Q − − − + + − − SF3B1 110693 c.1866G > Tp.E622D − − − − − − − SF3B1 110695 c.1874G > T p.R625L − − − − − − −SF3B1 131560 c.1984C > G p.H662D − − − − − − − SF3B1 130416 c.1986C > Ap.H662Q − − − − − − − SF3B1 110692 c.1986C > G p.H662Q − − − − − − −SF3B1 110694 c.1996A > G p.K666E − − − − − − − SRSF2 98000028 c.284C > AP95H − + + + + + − SRSF2 98000029 c.284C > T P95L − + + + + − − SRSF298000030 c.284C > G P95R − + + + + + − TET2 41644 c.1648C > T p.R550*− + + − − − − TET2 43417 c.2746C > T p.Q916* − − − − − − − TP53 10648c.524G > A p.R175H − − − − − − − TP53 10662 c.743G > A p.R248Q − + + + −− − TP53 10660 c.818G > A p.R273H − − − − + − − TP53 10656 c.742C > Tp.R248W − − − − − − − TP53 10659 c.817C > T p.R273C − − − − + − − TP5310704 c.844C > T p.R282W − − − − − − − TP53 10817 c.747G > T p.R249S− + + + + + − TP53 6932 c.733G > A p.G245S − − − − + − − TP53 10758c.659A > G p.Y220C − + + − + − − TP53 10654 c.637C > T p.R213* − − − − −− − TP53 10670 c.469G > T p.V157F − − − + − − − TP53 10705 c.586C > Tp.R196* − − − + − + − TP53 10645 c.527G > T p.C176F − − + + − + − TP5310889 c.536A > G p.H179R − + + + − + − TP53 10808 c.488A > G p.Y163C− + + + − − − TP53 10722 c.853G > A p.E285K − + − − − + − TP53 43606c.734G > A p.G245D − + + + + − − TP53 10779 c.818G > T p.R273L− + + + + + − TP53 10725 c.701A > G p.Y234C + + + + − + − U2AF3598000031 c.470A > C Q157P + + + + − + − U2AF35 98000032 c.470A > G Q157R− + + + − + − U2AF35 98000033 c.101C > T S34F − − − − − − − U2AF3598000034 c.101C > A S34Y + + + + − + − DNMT3A 99000100 copy_numbercopy_number − − − − − − −

Sample was fresh and sorted prior to genomic DNA isolation. According tothe manufacturer's analysis description: the raw CT for a given mutationassay in a test sample is compared with a predefined CT cutoff. Based onthe difference, the mutation can be considered as “Present” (+),“Borderline” (−/+), or “Absent” (−).

Recognized functional properties of MDSC include suppression of antigenstimulated or CD3 stimulated T cell proliferation and interferon-gamma(IFN-γ) production (Gabrilovich, D. I., et al. 2009. Nat Rev Immunol9:162-174; Ostrand-Rosenberg, S., et al. 2009. J Immunol 182:4499-4506).T cells purified from the BM of MDS patients showed reduced T cellproliferation (FIG. 1C) and IFN-γ production (FIG. 1D) after co-culturewith autologous MDS-MDSC, demonstrating the expected suppressiveactivity of these cells. To further validate these findings, MDSCs weredepleted from MDS BM specimens prior to anti-CD3/anti-CD28 stimulationand then the MDSC were added back to the control group. MDSC-depletionsignificantly improved T cell responses compared to theMDSC-supplemented group (FIG. 1E), thereby linking the observed impairedT cell responsiveness to the actions of BM-derived MDSCs. In addition,suppressive MDS-MDSC overproduced suppressive cytokines such as IL-β andTGF-β (FIG. 1F & G), as well as nitric oxide (NO) and arginase comparedto MDSCs isolated from healthy donors (FIG. 1H & I). Collectively, thesedata demonstrate that LIN⁻HLA-DR⁻CD33⁺ MDSC are a unique and functionalcellular subset supporting a pro-inflammatory microenvironment andimmune tolerance in the BM of MDS patients.

MDSC-mediated suppressive and cytotoxic effector functions requiredirect contact with target cells. One mechanism utilized by cytotoxiceffectors is the mobilization of pore-forming granules and the releaseof caspase-activating effector proteases, such as granzyme B, to thesite of effector:target contact thereby inducing apoptosis of the targetcell (Chen, X., et al. 2008. Blood 113(14):3226-34). Since MDSC residein close proximity with hematopoietic progenitor cells (HPCs) that inMDS display an increased apoptotic rate, MDSC-mediated cytostaticactivity may contribute to HPC death. To address this, MDS-MDSC granulemobilization and release of granzyme B were examined using four-colorimmunofluorescence staining. MDS-MDSCs exhibited strong granzyme Bpolarization at the site of cell contact with CD235a⁺ (glycophorinA)/CD71⁺ autologous erythroid precursors (FIG. 1J). After 30 minutesincubation, the frequency of such effector-target conjugates in MDSpatient specimens was significantly higher (34%) than in samples fromhealthy donors (5% P<0.001, FIG. 1K & L). These cellular interactionsresulted in apoptosis of targeted erythroid precursors (FIG. 1M)demonstrating that in addition to known MDSC-mediated immune suppressivefunctions, there is an unrecognized MDSC-mediated hematopoieticsuppressive capacity. To corroborate this finding, the effects of MDSCon the proliferative capacity of HPCs were examined inMDS-MDSC-depleted, HSPC enriched, BM patient specimens in amethylcellulose colony formation assay. Burst-forming unit-erythroid(BFU-E) and colony forming unit-granulocyte/macrophage (CFU-GM) colonyformation was significantly higher in MDSC-depleted specimens comparedto both MDSC-supplemented and unsorted samples (FIG. 1N), demonstratingthat MDSC have a direct suppressive role on erythroid and myeloidprogenitor cell development.

Increased CD33 expression and signaling contribute to MDSC suppressivefunctions and hematopoietic impairment. MDSCs in humanscharacteristically express the surface transmembrane glycoprotein CD33,a Siglec 3 receptor that along with other members of this family, haveprominent roles in inflammation (Crocker, P. R., et al. 2007. Nat RevImmunol 7:255-266; Blasius, A. L., et al. 2006. Blood 107:2474-2476;Blasius, A. L., et al. 2006. Trends Immunol 27:255-260; Lajaunias, F.,et al. 2005. Eur J Immunol 35:243-251; Nutku, E., et al. 2003. Blood101:5014-5020; von Gunten, S., et al. 2008. Ann N Y Acad Sci 1143:61-82;Paul, S. P., et al. 2000. Blood 96:483-490; Ulyanova, T., et al. 2001. JBiol Chem 276:14451-14458; Avril, T., et al. 2004. J Immunol173:6841-6849; Ikehara, Y., et al. 2004. J Biol Chem 279:43117-43125).However, the involvement of CD33 in myelopoiesis remains unexplored.Hence, the relationship between CD33 membrane expression density onMDS-MDSC and their activation and maintenance in MDS BM wasinvestigated. These cells were found to robustly express CD33 at levelshigher than LIN-HLA-DR-CD33⁺ cells isolated from non-MDS-associatedcancer patients and healthy donor BM cells (FIG. 2A). To explore thefunctional consequences of CD33 engagement, CD33 was cross-linked inU937 cells (a human monocytic cell line with high CD33 expression),which triggered IL-10, TGF-β and VEGF secretion (FIG. 2B). To examinewhether CD33 can promote MDSC accumulation and/or activation, CD33 wasoverexpressed with an adenovirus vector in BM-MNC from healthy donors,which significantly suppressed myeloid cell development as evidenced byreduced expression of the maturation markers CD11c, CD80, and CCR7 (FIG.2C). To further establish the role of CD33 in MDSC-mediated BMsuppression, CD33 was knocked down in MDS-MDSC using a lentiviral vector(LV) containing CD33-specific-shRNA. Co-culture of autologous HPCs withCD33 shRNA-treated MDS-MDSCs resulted in a 2-3 fold increase in BFU-Eand CFU-GM colony recovery compared to those cultured with scrambledshRNA-treated and non-transduced MDS-MDSCs and healthy donor BM MDSCs(FIG. 2D). Moreover, production of IL-10, TGF-β, and arginase wasreduced in CD33 shRNA-treated MDS-MDSC compared to control cells (FIG.2E, F, G). Collectively, these data delineate a role for CD33 in MDSCactivation and expansion in MDS and directing hematopoietic impairment.

S100A9 is a native ligand for CD33. Although these investigations showedthat CD33/Siglec 3 is a key receptor involved in functional activationof MDSC, its native ligand was unknown. Therefore, to identify potentialligand(s) for this receptor, a chimeric fusion protein with theectodomain of CD33 and the Fe portion of human IgG (here on referred toas CD33-fusion) was produced. BM cell lysates from MDS patients wereimmunoprecipitated with the CD33-fusion followed by high throughputmass-spectrometric analysis on associated peptides. S100A9, aninflammatory signaling molecule known to activate MDSC, was among themost prominent protein bands identified (FIG. 3A). To confirm thespecificity of the binding of this DAMP to CD33 both S100A8 and S100A9transfectants were prepared in the rhabdomyosarcoma cell line SJCRH30,which lacks detectable expression of endogenous CD33, S100A8 or S100A9.The CD33-fusion (APC) stained only S100A9 transfected cells, but notS100A8 transfected cells, confirming binding specificity (FIG. 3B.Transfectants were stained with FITC and specific binding by theCD33-fusion was stained with APC, (nuclei were stained with DAPI).Direct binding of S100A9 to CD33 was confirmed further in a sandwichELISA where the capture antibody was anti-S100A9 (FIG. 3C) as well as bydot blot analysis of transfected cell lysates with CD33-fusion (FIG.3D), demonstrating the specificity of the interaction. To fullycorroborate the binding of this pair, a reverse immune-precipitation wasperformed on CD33 transfected cells showing that S100A9 co-precipitatedwith CD33 only in S100A9 co-transfected cells (FIG. 3E). To validateclinically the ligand specificity in MDS patients, the pull-down wascompared with CD33-fusion from healthy PBMC and BM as well as MDSpatients. As expected, the highest amount of S100A9 was precipitatedfrom the BM of MDS patient specimens (FIG. 3F). Next, to understand thekinetics of S100A9 and CD33 interactions, SJCRH30 cells were transfectedwith either vector or CD33 and incubated with recombinant human (rh)S100A9 tagged with DDK (DYKDDDDK epitope, same epitope as Flag). Thisresulted in a time-dependent increase in binding of rhS100A9 to stableSJCRH30-CD33 cells but no binding to vector transfected cells (FIG. 3G).To demonstrate the functionality of this ligation pair, rhS100A9 wasadded to SJCRH30-CD33 cells which triggered CD33 mediated up-regulationof IL10 and TGFβ expression (FIG. 3H and I). To confirm that rhS100A9can recapitulate these observation of the secretion of these cytokinesafter cross-linking CD33, the experiments were repeated in U937 cellswith rhS100A9 and an increase in production of both cytokines was againfound (FIG. 3J and K). Importantly, BM plasma concentration of S100A9was significantly increased in MDS patients compared to BM plasma fromhealthy donors (FIG. 3L). Moreover, the engagement of CD33 with rhS100A9in MDS-MDSC from patient BM resulted in a time-dependent co-localizationof this ligand/receptor pair in primary MDS-BM cells (FIG. 3M). It iswell recognized that CD33 signals through phosphorylation of ITIMs thatrecruit SHP-1 (Src homology region 2 domain-containing phosphatase-1).rhS100A9 ligation correspondingly increased the recruitment of SHP-1confirming that CD33 signals through its ITIM after S100A9 ligation inMDS-BM (FIG. 3N). In addition, directly treating CD33-transfectedSJCRH30 with the BM plasma of MDS patients triggered the recruitment ofSHP-1 to ITIM when compared to cells treated with the plasma of healthydonors, suggesting that increased secretion of S100A9 in the local BMmicroenvironment may have a role in the activation of CD33-ITIMsignaling (FIG. 3O).

S100A8/S100A9 engagement of CD33 triggers MDS-MDSC activation. Havingestablished S100A9 and CD33 as a functional ligand/receptor pair, therole of this interaction in replicating the functional responsesobserved with receptor crosslinking was explored. CD33 overexpressionwas again induced in healthy BM cells through adenovirus transfectionand their immunesuppressive properties with or without the addition ofrhS100A9 was studied. Forced expression of CD33 induced a parallelincrease in gene expression and secretion of the suppressive cytokines,IL-β and TGFβ (light grey bars, FIG. 4A-D), which was greatly increasedby the addition of rhS100A9 (black bars). Secretion of the suppressivecytokine TGFβ was only observed after rhS100A9 treatment of CD33transfected cells (FIG. 4D), accompanied by an increase in theexpression of NOS2 and ARG2, consistent with the suppressive cytokineprofile (FIG. 4E and F). FIG. 4G-H demonstrates the transfectionefficiency of the CD33 adenovirus at both the gene and proteinexpression level measured by QPCR and GFP by flow cytometry,respectively.

To further delineate the specific role of S100A9's ligation with CD33compared to its other ligands, RAGE, TLR4, CD33 as well as each pairwere blocked with antibodies before treating healthy BM cells withrhS100A9. These data show that blockade of CD33 reduced both IL-β andTGFβ production, while treatment with anti-RAGE and anti-TLR4 had nosignificant effects on IL-10 production but displayed modest suppressionof TGFβ secretion and expression (FIG. 4I and J). These findings suggestthat although CD33 plays a critical role in the secretion of bothcytokines, other receptors associated with local bone marrowinflammation may also have a contribution. To confirm that S100A9expression contributes to inflammation in the BM microenvironment,S100A8 and S100A9 were knocked down in MDS-MDSC using gene specificshRNAs (FIG. 4K). Given that S100A9 usually pairs with S100A8 as aheterodimer and are concomitantly regulated, it was not surprising thatthere was reciprocal changes in gene expression with down-regulation inexpression of the alternate protein. Importantly, reduction ofS100A8/S100A9 expression profoundly attenuated IL-β and TGF-β production(FIG. 4L and M) and rescued autologous BFU-E and CFU-GM colony formation(FIG. 4N). These data show that inflammation-associated S100A8/S100A9signaling plays a critical role in the activation of MDS-MDSC andsuppress normal hematopoiesis.

S100A9 transgenic mice recapitulate features of human MDS. Given thefindings that S100A8/S100A9 triggers CD33/Siglec 3 signaling and isinvolved in MDSC activation (Ehrchen, J. M., et al. 2009. J Leukoc Biol86:557-566; Vogl, T., et al. 2007. Nat Med 13:1042-1049; Cheng, P., etal. 2008. J Exp Med 205:2235-2249), S100A9 transgenic mice (S100A9Tg)were generated to study MDSC-associated inflammation (Cheng, P., et al.2008. J Exp Med 205:2235-2249). Since MDS is an age-associated disease,changes in the proportion of BM MDSC (Gr1⁺CD11b⁺ cells) with age wereanalyzed in S100A9Tg compared to S100A9 knockout (S100A9KO)(Manitz, M.P., et al. 2003. Mol Cell Biol 23:1034-1043) or wild-type (WT) mice at6, 18 or 24 weeks of age. This resulted in a marked age-dependentaccumulation of MDSC in the BM of S100A9Tg mice, but not in S100A9KO orWT mice, that reached its maximum by 24 weeks (FIG. 5A). Similarly, theproportion of MDSC in PBMC and spleen also increased with age (FIG. 5B).Although changes in the proportion of MDSC in the spleen wascomparatively less than in the BM, it was accompanied by a decrease inthe proportion of mature cells (FIGS. 5C and 5D), effectively increasingthe ratio of immature to mature cells in this hematopoietic organ.Functionally, only the MDSC from 24-week old S100A9Tg mice, but notS100A9KO or WT mice, significantly inhibited BFU-E (FIG. 5E) and CFU-GMformation, which was rescued after depletion of MDSC in the S100A9Tggroup (FIG. 5E). Importantly, as further evidence of the role of S100A9as an essential inflammatory factor regulating MDSC expansion andsuppressive activity, IL-β and TGF-β secretion was significantlyincreased in S100A9Tg mice compared to KO or wild-type animals (FIG.5F).

To evaluate the in vivo consequences of S100A9 over-expression onhematopoiesis, serial complete blood counts (CBC) were analyzed from WTand S100A9Tg mice at 6, 18 and 24 weeks of age. S100A9Tg mice developedprogressive multilineage cytopenias characterized by decreasinghemoglobin (Hgb), red blood cell (RBC) number, neutrophil and plateletcounts evident as early as 6 weeks of age. By 18 weeks, S100A9Tg miceexhibited severe anemia and thrombocytopenia with a greater than 22.0%decrease in RBC, 20.1% decrease in Hgb, and 77.8% decrease in platelets(Table 2). Histological examination of BM aspirates and biopsy sectionsfrom WT mice displayed normal morphology and cytological features (FIG.5G-J). In contrast, the BM of S100A9Tg mice was hyper-cellular (95%cellularity) accompanied by tri-lineage cytological dysplasia (FIG.5K-N). Megakaryocyte morphology recapitulated the dysplastic featurescharacteristic of human MDS, with a preponderance of mononuclear orhypolobated forms. Erythroid precursors displayed megaloblastoidmaturation with abnormal hemoglobination and occasional binucleation.Nuclear budding and bizarre mitotic forms were also apparent (FIG.5K-N). Cytopenias worsened with age and by 24 weeks S100A9Tg micedeveloped severe pan-cytopenia (detailed descriptions summarized in thefigure legend). Although this model closely replicates the inflammatorymilieu observed in human MDS, HSPC from S100A9Tg mice also express theS100A9 protein. Given that the provenance of this protein in human MDSis not known, cellular expression of S100A9 in MDS BM-MNC wasinvestigated by flow cytometry. S100A9 intracellular staining wasdetected in CD33⁺ cells, whereas CD34⁺ HSCs had no demonstrable S100A9expression (FIGS. 5O and 5P). S100A9 expression was not detected inother immune cells such as CD3⁺ lymphocytes, CD19⁺ B cells or CD56⁺ NKcells.

TABLE 2 Complete peripheral blood count of S100A9Tg and wt-mice 6 weeks18 weeks 24 weeks Test Units WT S100A9-Tg WT S100A9-Tg WT S100A9-Tg WBC10³/μl 5.4 ± 0.5 4.2 ± 1.5 6.0 ± 1.4 2.9 ± 0.2* 6.3 ± 0.8 3.0 ± 0.4* LYM10³/μl 3.9 ± 0.5 2.6 ± 1.2 4.7 ± 1.1 2.4 ± 0.1* 4.8 ± 0.7 2.5 ± 0.3*MONO 10³/μl 0.4 ± 0.1 0.3 ± 0.2 0.4 ± 0.1 0.2 ± 0.1* 0.4 ± 0.1 0.2 ±0.1* GRAN 10³/μl 1.1 ± 0.6 1.2 ± 1.2 0.9 ± 0.3 0.3 ± 0.2* 1.1 ± 0.4 0.3± 0.1** HCT % 48.1 ± 3.2 42.4 ± 2.1 45.7 ± 0.7 35.5 ± 3.0** 45.4 ± 3.532.1 ± 2.7** MCV fl 51.4 ± 1.6 49.8 ± 2.0 50.3 ± 0.5 50.0 ± 1.2 50.0 ±1.3 50.0 ± 1.6 RDWa fl 35.0 ± 0.7 33.2 ± 2.2 34.0 ± 1.3 32.5 ± 1.7 33.3± 1.4 32.2 ± 1.5 RDW % % 16.3 ± 0.7 15.9 ± 0.1 16.3 ± 0.5 15.5 ± 0.516.0 ± 0.5 15.4 ± 0.6 HGB g/dl 14.2 ± 0.8 13.0 ± 0.7 13.9 ± 0.3 11.1 ±0.7** 13.7 ± 0.6 10.3 ± 0.5** MCHC g/dl 29.5 ± 1.1 30.6 ± 1.3 30.4 ± 0.431.5 ± 0.9 30.3 ± 1.0 32.3 ± 1.1 MCH pg 15.1 ± 0.3 15.2 ± 0.1 15.3 ± 0.115.8 ± 0.3 15.2 ± 0.2 16.2 ± 0.4 RBC 10⁶/μl 9.4 ± 0.7 8.5 ± 0.4 9.1 ±0.1 7.1 ± 0.6** 9.1 ± 0.3 6.4 ± 0.2*** PLT 10³/μl 555.7 ± 96.6 412.0 ±124.0 431.3 ± 33.9 95.7 ± 35.0*** 437.0 ± 41.9 61.0 ± 23.5*** All dataare means ± SEM (n = 3-5 mice). Peripheral blood samples were preparedfrom both S100A9Tg and control (WT) mice in ages of 6, 18 and 24 weeksand analyzed on a Hema True Hematology Analyzer (Heska). *p < 0.05; **p< 0.01; ***p < 0.001 vs wt-mice

Analysis of the role of MDSC by adoptive transfer of enriched HSC fromS100A9Tg mice. To more accurately delineate the role of MDSC fromS100A9Tg mice in hematopoiesis, competitive adoptive transfer wasperformed of enriched HSCs into lethally irradiated (900cGy) femaleFVB/NJ mice with age-matched WT HSC, S100A9Tg HSC or an admixture (1:1ratio) of enriched BM HSCs from male mice. Using a male to female SRYgene expression PCR approach to monitor engraftment, all miceexperienced greater than 80% engraftment (FIG. 8 ). After engraftment(defined as WBC >3×10³ cells/uL in WT recipients at 8 weeks), recipientsof WT HSC had proportions of both BM derived Gr1⁺Cd11b⁺ and HSCs (FIG.6A and C) that were comparable to levels in un-transplanted WT mice(FIG. 5A). In contrast, adoptive transfer with S100A9Tg enriched HSCsgenerated a high proportion of GFP expressing Gr1⁺Cd11b⁺ MDSCs (FIG. 6B)accompanied by a reduced proportion of HSCs, findings analogous to ourobservations in older transgenic mice (FIG. 6C). However, mice thatreceived the admixed HSC population had a proportion of MDSCsapproaching that in WT adoptively transferred mice (˜30%). Notably,nearly 50% of MDSCs lacked GFP expression, indicating origination fromWT HSPCs (FIG. 6B), whereas the remaining GFP⁺ MDSC derived fromS100A9Tg donor cells. Although the total MDSC population did notincrease to the level observed in the S100A9Tg adoptively transferredmice, a decreased proportion of HSCs to levels found in micetransplanted with S100A9Tg donor cells was observed (FIG. 6C). Thesefindings indicate that the smaller population of activated MDSCs fromS100A9Tg donor cells had sufficient suppressive activity to yield acomparable impairment of hematopoietic integrity. These findings weresupported by sequential analyses of peripheral blood counts in whichmixed source transplant recipients had cytopenias that were intermediatein severity relative to those in mice receiving the S100A9Tg or WT donorcells (FIG. 6D, E, F). Interestingly, while mixed donor recipients hadthe same proportion of HSCs after engraftment as Tg transplanted mice,their WBC counts were higher than mice transplanted with S100A9Tg HSCand lower than levels in WT HSC recipients. Similarly, onset of anemiawas delayed in recipients of the mixed versus S100A9Tg donor cells.These findings suggest that normal HSC from WT mice are able topartially rescue hematopoiesis, but with time hematopoiesis issuppressed by accumulating MDSC derived from S100A9Tg donor cells.

To address whether S100A9 alone has direct effects on HSCs, BM CD34+(MDSC depleted, MDSC−) from MDS patient specimens were treated withrhS100A9 for 24 and 48 hours and assessed apoptosis by flow cytometry. Adecrease in the number of CD34⁺ HSPCs was observed after treatmentcompared to controls accompanied by a corresponding increase in theapoptotic fraction among surviving cells after 48 hours exposure (FIG.6G and H). In order to corroborate these findings, healthy bonemarrow-derived CD34⁺ cells (Lonza, Wakerfield) were cultured withrhS100A9 and a decrease in viable cells was again observed aftertreatment (50.7% viability in control cells versus 24.7% in rhS100A9treated cells, FIG. 6I). These findings suggest that S100A9 has a directapoptotic effect in human HSCs.

Forced maturation of MDSC restores hematopoiesis. To confirm theeffector role of MDSC and investigate the potential benefit of targetedsuppression of MDSC, S100A9Tg mice were treated with all-trans-retinoicacid (ATRA). ATRA induces MDSC differentiation into mature myeloid cellsand neutralizes ROS production, thereby extinguishing MDSC throughforced terminal differentiation (Nefedova, Y., et al. 2007. Cancer Res67:11021-11028; Mirza, N., et al. 2006. Cancer Res 66:9299-9307). Tothis end, it was examined whether ATRA would induce MDSC differentiationin S100A9Tg mice and improve hematopoiesis. S100A9Tg and WT mice weretreated with ATRA (250% g/200% t) or vehicle control orally for fiveconsecutive days. Two days after completion of the treatment, ATRAreduced the total number of MDSC while numbers in WT mice remained atbasal levels (FIG. 7A). Reductions in MDSC number in S100A9Tg mice werecoupled to an increase in mature cells following ATRA treatment (FIG.7B). Treatment of primary MDS BM specimens with ATRA also reduced invitro MDSC accumulation. Importantly, BM progenitor cultures fromATRA-treated S100A9Tg mice showed significantly improved BFU-E recoverycompared to vehicle-treated controls (FIG. 7C). Analysis of changes inperipheral blood counts showed that RBC, WBC and platelet countssignificantly increased compared to vehicle treated controls (FIG. 7D),indicating that terminal differentiation of MDSCs can restore effectivehematopoiesis.

Investigations showed that MDSC employ CD33-associated ITIMs to inhibittheir own cellular maturation (FIG. 3N and O). However, ITIM signals canbe over-ridden by stimulatory immunoreceptor tyrosine-based activationmotif (ITAM)-mediated signals (Lanier, L. L. 2005. Annu Rev Immunol23:225-274; Ravetch, J. V., et al. 2000. Science 290:84-89). SomeCD33-related receptors, such as certain Siglecs, lack ITIMs and insteadfunction as activating receptors. Mouse Siglec-H and human Siglec-14have been shown to interact with DAP12 (Blasius, A. L., et al. 2006.Blood 107:2474-2476; Blasius, A. L., et al. 2006. Trends Immunol27:255-260; Angata, T., et al. 2006. Faseb J 20:1964-1973; Lanier, L.L., et al. 1998. Nature 391:703-707), an ITAM-containing adaptor thatcan promote myeloid cell maturation (FIG. 9 ) and inhibit TLR-4activation (Turnbull, I. R., et al. 2007. Nat Rev Immunol 7:155-161).Rhe DAP12 gene expression of purified MDS-MDSC (n=5) was compared toage-matched healthy donor MDSC (n=5). DAP12 mRNA was significantly lowerin MDS-MDSC in all specimens tested (FIG. 7E). It was reasoned thatoverriding CD33-ITIM signaling via DAP12 would induce thedifferentiation of these immature myeloid cells and improvehematopoiesis. To test this, a constitutively active form of DAP12,named P23, was created and AD293 cells transfected with either GFP,WT-DAP12, or active-DAP12 P23 viral vectors. The results show that P23binds Syk kinase and activates downstream signaling in transduced AD293cells without external stimulation (FIG. 7F). Furthermore, P23 promotesprimary human DC maturation as demonstrated by up-regulation of CD80,CD83, and CCR7 antigens (FIG. 10 ) (Blasius, A. L., et al. 2006. Blood107:2474-2476; Blasius, A. L., et al. 2006. Trends Immunol 27:255-260).Based on these findings, it is possible that P23 could induce thematuration of MDS-MDSC and thereby prevent or disrupt hematopoieticsuppression. To test this, expression of the human monocytic andgranulocytic surface markers CD14 and CD15 were analyzed aftertransfection (Araki, H., et al. 2004. Blood 103:2973-2980) and resultsshowed that while WT-DAP12 transfection alone was sufficient to induceup-regulation of CD14 and CD15, P23 transfection induced even greaterexpression of both maturation markers (FIG. 7G). Moreover, P23 promotedthe maturation of primary MDS-MDSC as demonstrated by up-regulation ofCD80, CD83, and CCR7 maturation markers (FIG. 7H). Lastly, to testwhether DAP12-mediated MDSC maturation relieved suppression oferythropoiesis, MDS-MDSC were purified from seven MDS patients andinfected with control, WT-DAP12 or P23 lentiviral constructs. To assessthe suppressive function of the mature MDS-MDSCs on hematopoiesis,colony forming capacity was assessed after culture of infected cellswith autologous, MDSC-depleted BM cells. P23-infected MDS-MDSCco-cultures yielded significantly higher colony numbers than controlviral vector or WT-DAP12-infected MDS-MDSC co-cultures (FIG. 7I). Thesefindings indicate that DAP12 overrides CD33-associated ITIM signaling tostimulate MDSC maturation, and reverse the suppressive effects on HPCcolony forming capacity.

Discussion

Inflammatory stimuli within the BM microenvironment are recognized asimportant biological signals stimulating progenitor cell proliferationand apoptosis in MDS. A recent population-based study extended thisfurther by demonstrating a strong linkage between chronic immunestimulation and MDS predisposition (Kristinsson, S. Y., et al. 2011. JClin Oncol 29:2897-2903). Definitive evidence that niche intrinsicabnormalities per se can alone account for development of MDS in a cellnon-autonomous fashion are limited. Raaijmakers and colleagues showedthat selective osteo-progenitor dysfunction caused by deletion of Dicer1in the mesenchymal component of the BM microenvironment was sufficientto perturb hematopoiesis and lead to development of myeloid dysplasia,followed by secondary emergence of myeloid-restricted geneticabnormalities (Raaijmakers, M. H., et al. 2010. Nature 464:852-857).

The disclosed studies show that Lin⁻rHLA-DR⁻CD33⁺ MDSC accumulate in theBM of MDS patients, derive from a population that is distinct from theneoplastic clone, and serve as cellular effectors that suppresshematopoiesis, promote T cell tolerance and serve as a key source ofmyelosuppressive and inflammatory molecules such as IL-10, TGF-β, NO,and arginase. Using multiple biological and biochemical approaches, itwas shown that S100A9, also known as migration inhibitory factor-relatedprotein 14 (MRP-14) or calgranulin-B, can serve as an endogenous nativeligand for CD33/Siglec 3. Furthermore, forced expansion of MDSC byover-expression of the S100A9 in transgenic mice initiates developmentof hematologic features that phenocopy human MDS, specificallyprogressive age-dependent ineffective and dysplastic hematopoiesis.These findings indicate that expansion of a single cellular constituentof the BM microenvironment is sufficient to foster neoplastic change inheterologous myeloid progenitors through niche-conducive oncogenesis.The time dependent accumulation of MDSC in the transgenic mouse modelparallels recent human findings that MDSCs expand with age accompaniedby rising serum levels of proinflammatory cytokines (e.g., TNF-α, IL-6,and IL-β), providing evidence that such senescence dependent changesdriving MDSC expansion may play an important role in the age dependentpathogenesis of MDS (Verschoor CP, et al. 2013. J Leukoc Biol93(4):633-7). Importantly, the LIN⁻HLA-DR⁻CD33⁺ phenotype did not aloneconfer suppressor cell function, as evidenced by lack ofLIN⁻HLA-DR⁻CD33⁺ MDSC suppression from age-matched healthy donors ornon-MDS cancer patients. The disclosed studies demonstrate the necessityfor activation of innate immune signaling and generation ofpro-inflammatory molecules, such as S100A9, for the induction ofMDS-MDSC-mediated suppressor function. Furthermore, the disclosedfinding that primary human MDS-MDSC lack molecular genetic abnormalitiesintrinsic to the malignant clone indicate that MDSC derive fromnon-neoplastic HSC, and that MDSC activation and expansion likelyprecedes emergence of genetically distinct MDS clones.

CD33, a Siglec receptor expressed by many immune cells including MDSC,is shown herein to be markedly over-expressed by MDS-MDSC, and thisreceptor is shown to control the suppressive functions of MDS-MDSCthrough disruption of ITIM-mediated signaling. Additionally, thedisclosed findings that rhS100A9 directly triggers apoptosis in humanHPCs, indicates that this ligand exerts dual roles in the promotion ofineffective hematopoiesis that involve both cellular (MDSC) and humoralmechanisms (CD33, TLR4). Moreover, cellular response to CD33 ligandengagement appears cell type specific, i.e., apoptosis in HPC versusactivation and proliferation in MDSC. Compensatory regeneration withinthe myeloid compartment could account for the increased proliferativeindex observed in MDS (Raza et. al. 1995. Blood 86(1):268-276) andpreferential myeloid skewing that occurs with age. Over-expression ofCD33 may therefore impair maturation signals from ITAM associatedreceptors that is critical to expansion of immature MDSC (FIG. 7G andH). Consistent with this, shRNA silencing of CD33 reducedmyelosuppressive cytokine elaboration, and importantly, restoredhematopoietic progenitor colony forming capacity. Moreover,constitutively active DAP12 signaling was sufficient to overrideCD33-ITIM inhibition and induce MDSC differentiation, which restorederythropoiesis upon active-DAP12 transfection into MDS-MDSC. Moreimportantly, DAP12 activation can inhibit TLR-mediated signalingpathways, which may also play a role in the inflammation-mediated BMsuppression (Turnbull, I. R., et al. 2007. Nat Rev Immunol 7:155-161;Hamerman, J. A., et al. 2006. J Immunol 177:2051-2055).

Mounting evidence implicates activation of innate immune signaling inthe pathogenesis and biologic features of human MDS (Hofmann, W. K., etal. 2002. Blood 100:3553-3560; Gondek, L. P., et al. 2008. Blood111:1534-1542; Starczynowski, D. T., et al. 2008. Blood 112:3412-3424).In del(5q) MDS, allelic deletion of miR-145 and miR-146a results inde-repression of the respective targets, TIRAP and TRAF6. Of particularimportance, Starcznowski and colleagues showed that knockdown of thesespecific miRs or over-expression of TRAF6 in murine HSPC recapitulatedhematologic features of del(5q) MDS in a transplant model through bothcell-autonomous and non-autonomous mechanisms involving interleukin-6(Starczynowski, D. T., et al. 2010 Nat Med 16:49-58; Starczynowski, D.T., et al. 2010. Hematol Oncol Clin North Am 24:343-359). The disclosedstudies show that the heterodimeric DAMP S100A8/S100A9, specificallyreleased at BM inflammatory sites, is not only aberrantly expressed inMDS but serves as a native ligand for CD33. This finding providesevidence that S100 proteins contribute directly to MDS pathogenesisthrough microenvironment-directed, cell non-autonomous mechanismsinvolving MDSC (Ehrchen, J. M., et al. 2009. J Leukoc Biol 86:557-566;Viemann, D., et al. 2007. Blood 109:2453-2460). Moreover, TLR activationsuppresses osteoblast differentiation, while instructing myeloidcommitment in HSC (Bandow, K., et al. 2010. Biochem Biophys Res Commun402:755-761; De Luca, K., et al. 2009. Leukemia 23:2063-2074). Prolongedactivation of innate immune signaling with age, therefore, may disruptthe BM endosteal niche supporting maintenance of hematopoietic stemcells, and favor translocation of myeloid progenitors to an angiogenicniche characteristic of MDS (Bellamy, W. T., et al. 2001. Blood97:1427-1434). This is supported by the disclosed findings of agedependent development of cytopenias with emergence of dysplasticcytological features in the S100A9Tg mice. More importantly, thecompetitive transplant experiments showed that admixture of S100A9Tgwith WT donor HSCs delays, albeit with time still impairs hematopoiesis.

The disclosed findings support a model for MDS pathogenesis in whichsustained activation of innate immune signaling in the BMmicroenvironment creates a permissive inflammatory milieu that issufficient for development of myelodysplasia. Cell autonomous neoplastichematopoietic progenitors may emerge following acquisition of secondarygenetic abnormalities in the myeloid compartment. S100A9Tg mice simulatehuman MDS and can serve as an in vivo model to study MDS pathogenesisand development of novel therapeutics. Nevertheless, therapeuticinterventions that promote MDSC maturation may have remitting potentialwhen applied early in the disease course.

Example 2

Active caspase-1 (Table 3) and IL-β generation (Table 4) were assessedin four populations: stem cells (CD34+CD38−), progenitors (CD34+CD38+),erythroids (CD71+), and myeloid cells (CD33+). Mean fluorescentintensity (MFJ) values are shown in Table 3.

TABLE 3 Treatment with CD33-IgG decreases active caspase-1 generation inan MDS specimen. Stem Cells Progenitors Erythroids Myeloids AncestryLIVE/CD34+/STEM LIVE/CD34+/PROGENITORS LIVE/CD71+ LIVE/CD33+ SubsetGeom. Mean Geom. Mean Geom. Mean Geom. Mean Value Type <FITC-A> <FITC-A><FITC-A> <FITC-A> For FAM FLICA FAM FLICA FAM FLICA FAM FLICA UNSTAINED 731  5760  189  3908 PLASMA ONLY 7062 61966 4170 44708 1 μg IgG 431638250 2836 37161 0.1 μg CD33-IgG 5232 43019 3339 40221 0.5 μg CD33-1gG5015 48556 3135 41516 1 μg CD33-1gG 3444 24089 2247 19533

TABLE 4 Treatment with CD33-IgG decreases IL-1β generation in an MDSspecimen. Ancestry LIVE/CD34+/STEM LIVE/CD34+/PROGENITORS LIVE/CD71+LIVE/CD33+ Subset Geom. Mean Geom. Mean Geom. Mean Geom. Mean Value Type<PE-A> <PE-A> <PE-A> <PE-A> For IL-1b-PE IL-1b-PE IL-1b-PE IL-1b-PEUNSTAINED 1457  25484  45.5 9979 PLASMA ONLY 201 9682 56.6 8884 1 μg IgG126 6667 15.5 8051 0.1 μg CD33-IgG 191 6380 48.2 8472 0.5 μg CD33-1gG151 10343  24.4 9073 l μg CD33-1gG 119 3092 12.6 3766

As shown in FIG. 12 , fold change of active caspase-1 and IL-βgeneration normalized to plasma treated control in four cellpopulations. Active caspase-1 and IL-β generation were assessed in fourpopulations by flow cytometry after treatment with CD33-IgG: stem cells(CD34+CD38−), progenitors (CD34+CD38+), erythroids (CD71+), and myeloidcells (CD33+). Fold change of active caspase-1 MFI (FIG. 12A) and IL-βgeneration (FIG. 12B).

As shown in FIG. 13 , fold change of active caspase-1 and IL-βgeneration normalized to plasma treated control in four cellpopulations. Active caspase-1 and IL-β generation were assessed in fourpopulations by flow cytometry after treatment with a related pathwayinhibitor: stem cells (CD34+CD38−), progenitors (CD34+CD38+), erythroids(CD71+), and myeloid cells (CD33+). Fold change of active caspase-1 MFI(FIG. 13A) and IL-β generation (FIG. 13B).

Active caspase-1 and IL-β generation were assessed in four populations:stem cells (CD34+CD38−), progenitors (CD34+CD38+), erythroids (CD71+),and myeloid cells (CD33+). Mean fluorescent intensity (MFI) values arefound in the Tables 3 and 4.

As shown in FIG. 14 , neutralization of plasma S100A9 by CD33 Chimeratrap enhances colony forming capacity in MDS patient specimens. FIGS.14A to 14C show erythroid burst-forming units (BFU-E) (FIG. 14B),multipotential colony forming units (CFU-GEMM) (FIG. 14A), andgranulocyte/macrophage colony forming units (CFU-GM) (FIG. 14C) in MDSpatient specimens treated with IgG, plasma, or 0.1, 0.5, or 1. μg ofCD33-IgG chimeric trap.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed invention belongs. Publications cited herein andthe materials for which they are cited are specifically incorporated byreference.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

What is claimed is:
 1. A recombinant fusion protein, comprising: (a) atleast two S100A9-binding moieties selected from the group consisting ofan extracellular domain of human CD33, an extracellular domain of tolllike receptor 4 (TLR4), and an extracellular domain of Receptor forAdvanced Glycation End Products (RAGE); and (b) an immunoglobulin Fcregion.
 2. The fusion protein of claim 1, comprising a formula selectedfrom the group consisting of: eCD33-eTLR4-Fc, eCD33-eRAGE-Fc,eTLR4-eRAGE-Fc, eRAGE-eTLR4-Fe, eCD33-eTLR4-eRAGE-Fe,eCD33-eRAGE-eTLR4-Fe, eTLR4-eCD33-eRAGE-Fe, eRAGE-eCD33-eTLR4-Fc,eTLR4-eRAGE-eCD33-Fc, and eRAGE-eTLR4-eCD33-Fc, wherein “eCD33”comprises the extracellular domain of human CD33, wherein “eTLR4”comprises the extracellular domain of TLR4, wherein “eRAGE” comprisesthe extracellular domain of RAGE, wherein “Fe” comprises theimmunoglobulin Fe region, and wherein “−” consists of a peptide linkeror a peptide bond.
 3. The fusion protein of claim 1 or 2, furthercomprising a biotin acceptor peptide that can be biotinylated withbiotin ligase (BirA) in the presence of biotin and ATP.
 4. A recombinantfusion protein, comprising: (a) an S100A9-binding moiety selected fromthe group consisting of an extracellular domain of human CD33, anextracellular domain of toll like receptor 4 (TLR4), and anextracellular domain of Receptor for Advanced Glycation End Products(RAGE); (b) a biotin acceptor peptide that can be biotinylated withbiotin ligase (BirA) in the presence of biotin and ATP; and (c) animmunoglobulin Fc region.
 5. The fusion protein of claim 4, comprising aformula selected from the group consisting of: eCD33-Fc-Avi,eTLR4-Fc-Avi, eRAGE-Fc-Avi, eCD33-eTLR4-Fc-Avi, eCD33-eRAGE-Fc-Avi,eTLR4-eRAGE-Fc-Avi, eRAGE-eTLR4-Fc-Avi, eCD33-eTLR4-eRAGE-Fc-Avi,eCD33-eRAGE-eTLR4-Fc-Avi, eTLR4-eCD33-eRAGE-Fc-Avi,eRAGE-eCD33-eTLR4-Fc-Avi, eTLR4-eRAGE-eCD33-Fe-Avi, andeRAGE-eTLR4-eCD33-Fc-Avi wherein “eCD33” comprises the extracellulardomain of human CD33, wherein “eTLR4” comprises the extracellular domainof TLR4, wherein “eRAGE” comprises the extracellular domain of RAGE,wherein “Fe” comprises the immunoglobulin Fe region, wherein “Avi”comprises an optional biotin acceptor peptide that can be biotinylatedwith biotin ligase (BirA) in the presence of biotin and ATP, and wherein“−” consists of a peptide linker or a peptide bond.
 6. The fusionprotein of any one of claims 3 to 5, wherein the biotin acceptor peptidecomprises the amino acid sequence SEQ ID NO:7, or an amino acid sequencehaving at least 90% identity to SEQ ID NO:7.
 7. The fusion protein ofany one of claims 1 to 6, wherein the extracellular domain of human CD33comprises only the variable region.
 8. A recombinant fusion protein,consisting essentially of: (a) the variable extracellular domain ofhuman CD33; and (b) an immunoglobulin Fc region.
 9. The fusion proteinof any one of claims 1 to 8, wherein the extracellular domain of humanCD33 comprises the amino acid sequence SEQ ID NO:1, or an amino acidsequence having at least 90% identity to SEQ ID NO:1.
 10. The fusionprotein of claim 9, wherein the extracellular domain of human CD33comprises the amino acid sequence SEQ ID NO:2.
 11. The fusion protein ofany one of claims 7 to 8, wherein the variable region of human CD33comprises the amino acid sequence SEQ ID NO:3, or an amino acid sequencehaving at least 90% identity to SEQ ID NO:3.
 12. The fusion protein ofclaim 11, wherein the variable region of human CD33 comprises the aminoacid sequence SEQ ID NO:4.
 13. The fusion protein of any one of claims 1to 12, wherein the extracellular domain of human TLR4 comprises theamino acid sequence SEQ ID NO:5, or an amino acid sequence having atleast 90% identity to SEQ ID NO:5.
 14. The fusion protein of any one ofclaims 1 to 13, wherein the extracellular domain of RAGE comprises theamino acid sequence SEQ ID NO:6, or an amino acid sequence having atleast 90% identity to SEQ ID NO:6.
 15. A multimeric complex comprisingtwo or more fusion proteins of any one of claims 1 to 14 conjugated to acore molecule or particle.
 16. The multimeric complex of claim 15,wherein the core molecule is streptavidin, wherein the two or morefusion proteins are biotinylated.
 17. The multimeric complex of claim15, wherein the core molecule is a liposome comprising antibodies thatspecifically bind the two or more fusion proteins.
 18. The multimericcomplex of claim 17, wherein the antibodies specifically bind the biotinacceptor peptide.
 19. The multimeric complex of any one of claims 15 to18, comprising from 2 to 5 fusion proteins conjugated to the coremolecule or particle.
 20. A method for treating a disease or conditioncaused or exacerbated by S100A9 activity, comprising administering to asubject in need thereof a composition comprising the fusion protein ofany one of claims 1 to 18 or the multimeric complex of any one of claims15 to
 19. 21. The method of claim 20, wherein the method comprisestreating an infection, sepsis, or a combination thereof in the patient.22. The method of claim 20, wherein the method comprises treating anautoimmune disease in the patient.
 23. The method of claim 20, whereinthe method comprises treating rheumatoid arthritis in the patient. 24.The method of claim 20, wherein the method comprises treating a cancerin the patient.
 25. The method of claim 20, wherein the method comprisestreating a myelodysplastic syndrome (MDS) in the subject.
 26. A methodfor treating myelodysplastic syndrome (MDS) in a subject, comprisingadministering to the subject a therapeutically effective amount of acomposition comprising a soluble CD33 fusion protein that binds andsequesters S100A9.
 27. A method for identifying an agent for treatingmyelodysplastic syndromes (MDS), comprising screening candidate agentsfor the ability to prevent S100A9 binding to CD33.